thirty-fourth annual meeting february 18-22, 1990 ...asher/homepage/spec_pdf... · invasion and...

SPECTROSCOPIC STUDIES (EPR, NMR)

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APPLICATIONS OF MAGNETICALLY COUPLEDRELAXATION SPECTROSCOPY TO UNDERSTANDINGMAGNETIC RELAXATION IN TISSUES ANDPROTEIN SYSTEMS. Robert G. Bryant,Biophysics Department, University ofRochester, Rochester, NY 14642.

Coupled magnetic relaxation is wellknown and forms the basis for solution ofmacromolecular structures in solutionphase experiments. However, the fullpractical and theoretical implications ofthis coupling have been overlooked inattempts to understand the behavior ofwater at interfaces. New experimentsthat exploit this magnetic relaxationcoupling permit direct characterizationof the proton spectrum of the solidcomponents while observing the narrowliquid components. The analysis andextension of this experiment provides thefundamental basis for a quantitativeunderstanding of the magnetic fielddependence of proton relaxation in hetero-geneous systems. The consequences have aprofound impact on the interpretation ofmagnetic relaxation in other liquidsystems including the controversial caseof protein solutions.

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31pNMR METABOLIC IMAGES OFTHE ARM.T.R. Brown, S.J. Nelson, J.S. Taylor, J. Murphy-Boesch, D.B. Vigneron, Dept. ofNMR and MedicalSpectroscopy, Fox Chase Cancer Center, Phila., PA

Measuring changes in metabolism within specificmuscle groups requires precise localization andcorrelation with anatomical features. Chemical shiftimaging (CSI) produces spectra from well defined voxelswhich can be mapped directly onto corresponding protonimages. To analyze this CSI data one must first quantifyindividual spectra and estinate metaboliteconcentradons. We have developed automaticquantification procedures and applied them to extractimages of the spatial distribution of phosphorousmetabolites in the ann. A single tum 125 mm diametersurface coil was placed around the arn and 2D-CSIdatasets collected with spatial resolution as low as 5 mm.Localization perpendicular to the plane of the coil wasapproximately 80 mm. Metabolic images of Pi, PCr, y-ATP, a-ATP and 1-ATP showed excellent correlationwith the antomy obtained from proton images. Dataacquisition times as short as 1' were used to followchanges in the 3lp metabolites during exercise andrecovery. Images of the metabolites showed changes inPCr, Pi and pH were localized to specific muscle groups:the flexor digitorum superficialis, the flexor digitorumprofundis and the extensores carpi radialis.

Biophysical Journal voL 57, 1990 43a

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STUDIES OF BIOLOGICALLY ACTIVE PROTEINFRAGMENT CONFORMATIONS USING 2D NMR METHODSJ.E. Furstenau*, J.A. Starkey* H.E. Hamm+,P.A. Hargrave# and E.A. Dratz', *MT StateUniv., #Univ. Ill Med Center, #univ. Fl(sponsored by A. Jesaitis).

Biological control and/or adhesionsystems often depend on interactionsbetween proteins that cannot becrystallized and/or that tumble too slowlyto allow structural studies by solutionNMR. In an number of interesting cases,synthetic peptide fragments of one proteincause the full biological activity in theother member of the interacting pair.

We are studying solution conformations(using primarily ROESY and TOCSY) andreceptor bound conformations (usingprimarily transferred NOESY) of severalsystems. Laminin peptide 11, which bindsto the laminin receptor on endothelialcells and inhibits metastatic tumorinvasion and retinal rod GTP-bindingprotein (transducin) Ac-311-329 and 340-350segments, which bind to light-activatedrhodopsin and hold it in metarhodopsin II(the form that binds to and activates thefull GTP-binding protein). (Support:Tobacco Res. Council and MT Center forExcellence in Biotechnology.)

M.Pos44NMR VISIBILITY OF SODIUM

IN RAT MUSCLER. Buist, R. Deslauriers, J.K. Saunders,G.W. Mainwood National Research Councilof Canada, Ottawa, Canada, KlA OR6

The NMR visibility of the Na-23nucleus has been debated for many years.Theoretically, it is possible that only40% of the signal is observable.

We have used the ratio method ofThulborn and Ackerman [1] to quantitatetotal Na levels in vivo in rat leg (n=6)and in excised rat gastrocnemius muscle(n=4). At the end of each NMR experiment,samples were extracted for measurement ofNa by flame photometry. Comparison of thetwo methods allows determination of NMRvisibility of Na-23. We have found morethan 80% of the Na to be visible in bothsample types.

Na-23 T2 relaxation times in intactrat leg are biexponential with values of4.4ms (40%) and 37ms (60%). The Tl valueis 51ms. These values compare to NaCl inaqueous solution for which Tl=T2=55ms.[1] K.R. Thulborn and J.J.H. Ackerman.Journal of Magnetic Resonance, 55: 357-371, (1983).This work was supported by the MedicalResearch Council of Canada (MT-625).

44a Biophysical Journal voL 57, 1990

M-Pos4519 F NMR ANALYSIS OF TFIFLURQRETHA-NOL METABO LITESE3 IN RAT URlNEBarry S. Selinsky and Dariee M.

Rusnyiak, Department of Chemistr-J.,Villanova University, Vi'llanova DA19085

Trifluoroethanol (TFEE) is a commonindustrial soivent and a knownmetabolite of the inlalation ares--thetic Fluoroxene (2,2,2-+tri-fluorcethyl vinyl ether). The uri-nary metabo 'ites of -ats dosed w.th0.21 9/kg (i.p.) trifluor'oethanolwere monitored using 19F tIMR spec-

troscopy. The predominant earIymetabolite observed in urine wasthe glucuronide conjugate of TFE,with concentrations approaching 30mM in urine collected eight hoursafter TFE injection. The oxidatior;products of TEE, triflucroacetalde-hyde and trifluoroacetate, are alsoobserved in rat urine following TFEtreatment. A fourth metabolite,yet to be identified, is alscfound; its excreticn parallels tineexcretion of trifluoroacetaldehn.de.This study demonstrates the uriIityof 19F NMR to examine the metabo.-issm of fluorinatec xenobiotics.

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NMR AND FLUORESCENCE OF 5-FLUORO-TRP-MELITTIN. Arthur J.Weavert, Marvin D. Kemple*, and Franklyn G.Prendergast§, tHoward Hughes Med. Inst., UT$Qlouthwestern, Dallas, TX 75235, *Dept. ofPhysics, IUPUI, Indianapolis, IN 46205, §Dept.of Biochem. and Mol. Biol., Mayo Foundation,Rochester, MN 55905.

An analog of the cytolytic peptide melittinwas synthesized with 5-fluoro-tryptophan atposition- 19 and 13Ca-glycine at position- 12.Column-chromatography experiments indicate thatthis peptide forms a NaCl-induced tetamer.NMR chemical shift measurements of 13Ca-gly-12 were consistent with tetrer formation. The19F-NMR signal from trp-19 registered a chemicalshift change of 0.9 ppm upon formation of theputative tetramer in aqueous solution. Chemicalshift changes of the 19F resonance induced byD20 are considered to be indicative of solventexposure of the residue in question. For themonomer a solvent-induced shift identical to thatof free 5-fluoro-trp was observed while for thetetramer no shift was seen implying completesolvent exposure of trp-19 in the former and littleexposure in the latter. Properties of this peptideunder vanrous solvent conditions as monitored byNMR and fluorescence will be described.Supported in part by N1486K0521 from ONR.


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TRNOE STUDY OF THE CONFORMATION OFMGATP BOUND TO NUCLEOTIDYL ANDPHOSPHORYL TRANSFER ENZYMES S. B.Landy, K. B. Lipkowitz, B.D. Nageswara RaoIUPUI, Indpls, In., 46205, and P. Plateau, EcolePolytechnique, France.

The conformations of MgATP bound to anucleotidyl transfer enzyme, methionyl tRNAsyntetase, and a phophoryl transfer enzyme,pyuvate kinase, were studied by 1H NMRtransferred NOE (TRNOE) measurements.Selective inversion of chosen resonances wasaccomplished with a DANTE sequence NOEmeasurements were made at 8 delay times rangingfrom 20 ms to 500 ms. A full complement of tenNOE build-up curves, for each enzyme complex,was analyzed by using the complete relaxation-matrix method modified to include exchangebetween bound and free substrate. Molecularmechanics computations were used to examine theenergetic implications of the NOE-determinedstructures. The final structures obtained forMgATP bound to the two enzymes were verysimilar to each other, with a 3' - endo sugar puckerand an anti confornation with a glycosidic torsionalangle (O'4 - C'l- Ng- C8) of 390 ± 40. (supportedin part by NSF DMB 8608185 and NIH GM43966)

M-Pos48THE BEHAVIOR OF HALOTHANE IN RABBITTISSUES AS MEASURED BY 19F SPIN-SPINRELAXATION TIMES.

Mitsue Miyazaki, Eugene Y. Shen,and Alice M. Wyrwicz. Department ofRadiology, University of Illinois at Chicago,Chicago, Illinois 60680.

The mechanism(s) of general anesthetic action:emains unknown. To gain further understandingof their behavior in tissues, 19F NMR spin-spinrelaxation times(TZ) of a fluorinated anesthetic,halothane, were measured using a CPMG pulsesequence. The T2 values for halothane in variousrabbit tissues given below, were measured atanmbient temperature('18°C) on a Bruker WP-200NMR spectrometer. Halothane in the braincerebrum and cerebellum gives similar 19F T2values of 3-4 msec. There is no distinction inthis value between the white and the grey matter.-This short T2 relaxation time is unique for thebrain tissue and indicates a considerably decreasedmobility for halothane. The other tissues havemuch longer T2 relaxation times: muscle, T2=391rimsec; adipose, T2=471 msec; infraorbital glands,TZ=334 rmsec. At higher inspired cocentratios ofhalothane an additional longer TZ(182 msec.)component is observed predominantly in thewhite matter of the brain. This suggests additionalbinding site(s) with higher mobility for halothanein the white matter.


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13C CP/MAS SOLID STATE NMR SPECTROSCOPYAND X-RAY STUDIES OF THE CARBOXYLATECOORDINATION OF PARAMAGNETIC SHIFT REAGENTSE.A. Adams, S.L. Helgerson, and E.A. Dratz,Chemistry Department, Montana StateUniversity, Bozeman, MT 59717.We have carried out model experiments toinvestigate the use of paramagnetic shiftreagents in solid state NMR to identifyside chains involved in calcium-bindingmembrane proteins. Paramagnetic (Eu+3,Sm+3, Pr+3) and diamagnetic (La+3, Ca+2)ions were complexed with ethylenediaminetetraacetic acid (EDTA). Eu+3 and Pr+3EDTA produced broad, weak carboxylate peaksshifted greater than 50 ppm. Sm+3 shiftsthe carboxylate resonances of EDTAdownfield by 12 ppm with only moderatebroadening. Sm+ also splits (5 ppm) thecarboxylate resonances of EDTA into twoequal peaks. X-ray diffraction shows thattwo pairs of carboxylate groups in Sm:EDTAare coordinated with different bondinggeometries. The main La:EDTA carboxylatepeak, is three carboxylate groups, and adownfield shoulder equal to a longer C-Odistance. (ONR K0278, NIH EY06913.)

M-Pos51SOLUTION STRUCTURES OF PEPTIDESCONTAINING THE RECEPTOR BINDINGSEQUENCE ARG-GLY-ASP. J.B. Vaughn, Jr.,A.S. Mildvan, and J.T. August. Depts. Biol. Chem.and Pharmacol., The Johns Hopkins MedicalSchool, Baltimore, MD 21205.

Two-dimensional proton NMR studies of asynthetic 14-residue peptide (YAVTGRGD-SPASSC), based on residues 80 to 92 offibronectin, were carried out at 600 MHz, 270,150 mM NaCI, and pH 5.9. Sequence-specificassignments of resonances were made by doublequantum filtered COSY, TOCSY, and NOESYspectra with jump-return solvent suppression. Theresults are consistent with a type II a-turn at theGRGD position, preceded and followed by long6-strands which approach each other, as shown byremote NOEs from Ha of Ala-2 and Val-3 to HNof Ala-11, and by several slowed NH exchangerates. However, a fully formed antiparallel#-sheet Is not detected. This secondary structurediffers in register from that of the simplerpentapeptide (GRGDS) which consists primarily ofa type II ,6-turn in the RGDS position (Cachau etal., these proceedings). Because of suchdifferences, it would be most appropriate to studythe conformations of receptor-bound RGD ligands.


M.PosSOI H NMR SPECTROSCOPY OF A SYNTHETIC23-MER FRAGMENT OF HUMAN RELAXIN. L^.HIazard, E. Bullesbach, C. Schwabe and T.Williams.Departments of Pharmacology and Biochemistry,Medical University of South Carolina, Charleston, SC29425-2251.

The human relaxin molecule, a peptide hormonewith 53 residues, is involved in the dilation of themammalian birth canal. The mechanism by which relaxinparticipates in this process is unclear, in part, due to alack of structural information. Relaxin appears to formaggregates in solution, making NMR analysis of its nativeconformation difficult. Because fragments of relaxinshow less tendency to aggregate, their solutionstructures may be more amenable to NMR analysis.Based on relaxin's disulfide homology to insulin, we haveselected and synthesized a 23-mer fragment of thehuman sequence which runs from residue 7 to 17 ofchain A and from residue 7 to 18 of chain B, includingboth the one intra-A-chain and one inter-chain disulfidebonds of the intact hormone.One dimensional 1H NMRspectroscopy of the 23-mer indicated that its singlehistidine titrates with an abnormally low pKa of 5.6. Fromtwo-dimensional spectroscopy, we have assigned nearlyall of the 1 H resonances of the the residues sequencespecifically. In order to understand the basis of thedepressed pKa of HisAl 2, we have used a series of lowresolution absolute value mode COSYs to determine thepH profile of most all side chain proton resonances.

M-Pos52MULTIPLE CONFORMATIONS OFWILD TYPEAND MUTANT STAPHYLOCOCCAL NUCLEASEAndrew Hinck, Stewart Loh, Jinfeng Wang,

Andrei Alexandrescu and John MareyDepartment of Biochemistry, Univ. Wisconsin-Madison

420 Henry Mall, Madison, WI 53706

We have investigated multiple conformations ofstaphylococcal nuclease by studying the His121 1iH82 and13CGI NMR signals in nuclease wild type (wt) and in themutant in which glycine 79 is changed to serine (G79S). Innucleasewt labeled with 13C 26% (ul) histidine, we find two1 j6Z/13 C82 crsspaksin the.1H( C) single bondco¢relation experiment Both signals are strongly upfieldshifted (4.3/116.5 and 4.8/121.0), presumably due to the ringcurrent effect of nearby Tyr91 , and are found in an intensityratio of 10:1. We attribute this heterogeneity at His1211H,2to the cis:trans isomrization about the Lys116 -pro117 peptidebond. In the nuclease mutant G79S this pattrn is revered.Based on their response of the His11HII signals of G79S toinhibitor binding, we assign the conformation about theLys116 -Pro117 peptide bond as cis in nuclease wt and trans innuclease G79S.

We also have observed two signals for His4 in theone-dimensional1HNMR spectrum. This heterogeneity isevident for both unligated nuclease and thenuclease Caeinhibitor terary complex in nuclease wt andmutants. In addition, mutant enzymes showing reversed N/N'ratios for the 111 of His121 , exhibit a wt-like distribution ofpeak intensites for His46. Thus, the heterogeneity we observeatHis46 does notappear to be due toiso riztion about theLys116-Pro117 pepide bond

46. Biophysical Joumnal vol 57, 199O

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1H-NNR STUDIES OF PSEUDOMONAS PUTIDAFERREDOXIN. Hong Cheng, Klaus Grohmann,William Sweeney. Dept. Chemistry, HunterCollege, 695 Park Ave., N.Y., NY 10021

eL piutia ferredoxin contains a 3Fe-4S and a 4Fe-4S cluster, and exhibitsextensive sequence homology to Azotobactervinelandii ferredoxin. The proton NMRspectrum exhibits six resolved downfieldresonances. All six resonances exhibit T,times S10 msec, indicating all arise fromprotons near an iron-sulfur center.Direct labeling studies have shown thatthe five most downfield resonances (31,25, 22, 18, and 16 ppm) arise from 0-

cysteinyl protons. Conflicting reportshave assigned the resonance at 16 ppm to aproton near the 3Fe center or near the 4Fecenter. Current studies suggest that thisresonance arises from a (-proton on acysteine bound to the 4Fe center but nearthe 3Fe center. The sixth resonance (10.5ppm) appears not to arise from a cysteinylproton. At temperatures 150 and k30O thisresonance splits into two peaks of approx-imately equal intensity. pH titrationstudies show that the three most downfieldresonances titrate with a pK close to 5.6.The resonances at 18 and 16 ppm titratewith pKs of 4.5 and 7, respectively.Supported by NSF grant DMB-84 16808.

M-Pos55NMR DETECTION OF CREATINE KINASE EXPRESSED IN.THE LIVER OF TRANSGENIC MICE: DETERMINATIONOF FREE ADP LEVEL.MJ. Brosrnn L. Chen-,J. Chenr, T.Van Dyke°, and A.P.Korebky Depts of Blo. Scl., Caoo gle Melon UrAv. andUliv. ofPttsbugh,Ptisbugh,PA 15213

ADP is an important regulator of a number ofprocesses, such as gluconeogenesis andoxidative phosphorylation. The equilibriumestablished by creatine kinase (CK) has beensuccessfully used to determine free ADP In muscle.broin, and heart. Liver locks CK, therefore. It hasbeen difficult to determine free ADP levels in liver Invivo. To remedy this we have expressed the Bisozyme of CK in the liver of transgenic mice. ADNA construct containing the liver speciffc controlregion from the tronsthyretin gene and the codingregion of the mouse brain CK gene was used togenerate three lines of transgenic mice. Thesemice have CK activity in liver ranging from 80-250gmoles/g wet wt at 25°C; this compares to <1limoles/g wet wt in control liver and 250 ±moles/gwet wt in mouse heart. 31p NMR spectra fromtransgenic liver contain a peak due tophosphocreatine which is absent from control liverspectra. Using the equilibrium established by CK afree ADP level of 0.059+.004 gmoles/g wet wt wasfound In transgenic liver. This value was constantover a broad range of CK activity and total livercreatine levels. These mice provide a valuabletool for understanding the role of ADP in hepaticenergy metabolism.

SPECTROSCOPIC STUDIES (EPRt NMR)

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PROTON-DETECTED NATURAL ABUNDANCE 13C NMRSPECTROSCOPY OF THE ZINC FINGER DNA-BINDINGDOMAIN XFIN-31. Arthur G. Palmer, III andPeter E. Wright, Department of MolecularBiology, Research Institute of ScrippsClinic, La Jolla, CA 92037

The increased sensitivity of proton-detected heteronuclear NMR spectroscopy,compared to conventional methods,facilitates the study of heteronuclei atnatural abundance in biomolecules.Recently, the zinc finger motif hasgarnered attention as a structural domainfor the specific recognition of DNAsequences. The DNA-binding domain Xfin-31is a 25-residue peptide, derived from theXenopus laevis protein Xfin, that binds asingle zinc atom and forms a compactglobular firucture in solution. Proton-detected C heterocorrelation and relayedheterocorrelation silctroscopies have beenused to assign the C spectrum of Xfin-31and two-dimensioyl proton-detectedmeasurements of C longitudinal andtransverse relaxation have been initiated.The results indicate the utility of proton-detected heteronuclear spectroscopy forspectral assignment, conformational andstructural analyses, and investigation ofrelaxation in biomolecules.

M-Pos56DIVALENT METAL BINDING TO CHEY:EFFECT ON PROTEIN CONFORMATION

AND PHOSPHATE BINDING

L. Kart, S. Roman§, M. Johnson$ & P. Matsumural;Department of Medicinal Chemistry &

§Department of Microbiology and ImmunologyUniversity of Illinois at Chicago

The interaction of the bacterial chemotaxis pro-tein, CheY, with ATP and a variety of other relatedmolecules has been studied in the presence and ab-sence of divalent metal ions. These studies have ledto the discovery of two conformations of ,the proteinin water. It is shown that, in the metal-bound eon-formation, CheY will bind to nucleotide phosphatesand phosphates in general; but in the metal-free con-formation the protein loses its affinity for phosphates.Comparison of double quantum filtered COSY spec-tra from the metal bound and muetal free forms ofthe protein indicates that two phelalanines and onethreonine are most significantly affected by the con-formational change involved. These same residuesare also indicated to be clo to the phosphate bind-ing site in NMR experiments with spin labeled ATP,suggesting that the loss of metal alters the conforma-tion of the phosphate binding site, thereby reducingbinding affinity for phosphates. The possible sig-nificance of metal binding on the phosphorylation &dephosphorylation of CheY is discussed.


MPos57

31p NMR STUDY OF THE EFFECT OF PB2+ON ATP SYNTHESIS RATE IN BONE CELLS.Dowd, T.L., Rosen, J.F. and Gupta,R.K. Albert Einstein College ofMedicine, New York, N.Y. 10461.

Evidence from various studiessuggests perturbations in cellularenergetics as the molecular basisfor Pb2+ toxicity. We haveinvestigated the effects of Pb2+ on

high energy phosphates and on theunidirectional rate of ATP synthesisin a continuously perfused ratosteoblastic bone cell line (ROS17/2.8) using 31p NMR and saturationtransfer. The mechanism of ATPy-*Pisaturation transfer was alsoinvestigated. The unimolecular rateconstant for ATP synthesis inuntreated cells was .25 + .04 sec-l.Upon incubation of the cells with 10sM Pb2+ no saturation transfer wasdetectable with our signal to noiseratio indicating a reduction in therate of ATP synthesis. In additionthere was a 34% decrease in ATP anda 39% decrease in PCr upon exposureto 10 AM Pb2+. This provides directevidence of Pb2+ induced alterationsin cellular energetics and mayexplain some previously observedfunctional impairments.

Biophysical Journal voL 57, 1990 478

48a Biophysical Joumal vol 57, 1990

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A 1W-RESONANCE RAMAN INVESTIGATION OFTRYPTOPHAN IN AZURIN. J. A. Sweeney,P. A. Harmon and S. A. Asher, Dept. ofChem., Univ. of Pittsburgh, C. M. Hutnikand A. G. Szabo, Div. of Biol. Sciences,Nat'l. Res. Council of Canada, Ottawa,

We investigated the role of Trp in theblue-copper protein azurin from Pseudomonasaeruginosa which serves as a redox carrierin electron transfer. Fluorescence studiesof holo and apo azurin indicate inter-actions between the Trp and the Cu whichdepends upon the Cu oxidation state. Weuse Raman Saturation Spectroscopy tomonitor repopulation of ground statemolecules subsequent to Raman excitation.The reciprocal of TRP ground state repopu-lation rates in holoazurin and apoazurinqualitatively agree with the fluorescencedata. TRP radical cation is not observed.The fast recovery of ground state Trp inholoazurin, indicates that the lifetime ofTRP radical must be less than 500 ps. Wealso examined the Raman cross sections ofTrp of Cu(I) and Cu(II), and guanidinehydrochloride denatured azurin. Theseresults reflect the hydrophobic Trpenvironment in the solvent-inaccessible3-barrel region of azurin and the highlypolar aqueous environment of the exposedTrp in the denatured protein.

MPosSONANOSECOND TRANSIENT ABSORPTION SPECTROSCOPYOF COENZYME B12: QUANTUM YIELDS AND SPECTRALDYNAMICS. Chen, E., Chemistry Department,Georgetown University, Washington, DC 20057.

For many B12 enzyme reactions, cleavageof the cobalt-carbon bond of adenosylcobala-min (Ado-Cbl) leads to the generation of aCo2+ ion and a free radical. Photolysis ofAdo-Cbl, which also leads to homolyticcleavage, may mimic the structure and dyna-mics of enzyme induced cleavage, providinga model system for enzymes. Therefore, wehave used laser photolysis and nanosecondtransient absorption spectroscopy with adouble diode array detector, in order tomeasure the quantum yields and spectral dy-namics of cobalamin compounds. The nano-second quantum yield of photolysis for Ado-Cbl is 0.23 + 0.04, higher than previouslyreported. "Base-off" B12, a more stable,acidified form of Ado-Cbl, has a fivefoldlower quantum yield at 0.045 + 0.015, show-ing that quantum yields reflect intrinsicbond strength. The minimal wavelengthdependence of the quantum yield for Ado-Cbl,demonstrates at least 50% efficiency incoupling of the corrin ring energy to thecobalt-carbon bond. In addition, these re-sults suggest that geminate recombinationmay play a significant role in the mecha-nism of homolytic cleavage.

SPECTROSCOPIC STUDEES (OTHER)M-Pos59FLUORESCENCE LIFETIME DISTRIBUTION OF TRYP-TOPHANYL RESIDUE AS FUNCTION OF POLYPEPTIDECHAIN LENGTH.Ettore BISMUTO & Gaetano IRACEDipartimento di Biochimica e BiofisicaUniversitA di Napoli, ITALY.

The aim of this report is to correlatethe emission lifetime distribution parame-ters to the polypeptide chain length. Inorder to exclude effects due to non-coval-ent interactions, we studied the fluorescence decays of protein fragments, in 6.0 Mguanidine, obtained from enzymatic digest-ion of tuna apomyoglobin,which contains a

single indole residue. Three fragments ofdifferent length were purified. Lifetimedeterminations were performed by frequencydomain fluorometry. The results show thatthe tryptophanyl lifetime distributionwidth of peptides containing more than 14residues is correlated to the chain lengthwhereas the emission decay of shortest pep-tides is similar to that observed forN-acetyltryptophanylamide.

M-PoS6lSTRUCTURES OF INTERMEDIATES OFCOENZYME B12 CATALYSIS. Sagi, I.,Wirt, M., Chen, E., Frisbie, S., andChance, M., Georgetown Univ., Dept.of Chemistry, Wash.,D.C.,20057.

The structures of the catalyticintermediates of adenosylcobalamin(Ado-Cbl) dependent enzyme systemsare not well understood and havebeen difficult to crystallize. UsingEXAFS spectroscopy, we have examinedthe structure of the Co*', Co+2, and"base-off" forms of B12. For the Co*2species, the average of the Co-Nequatorial distances of the corrinring are 1.87+0.01 A, and the Co-nitrogen (Co-Nd) distance to the di-methyl-benz-imidazole ligand is1.99+0.02 A. The large reduction inthe Co-Nd distance relative to Ado-Cbl, where Co-Nd is 2.24 A, is con-sistent with the cobalt ion movingout of the plane of the corrin ni-trogens, and suggests a mechanismfor cobalt-carbon bond homolysis.When the cobalt-carbon bond breaks,the Co-Nd bond becomes much strongerand electron density is reorientedaway from the breaking bond. We alsopresent the structures of Co*' andbase-off B12 and discuss theirsignificance to B12 catalysis.

SPECTROSCOPIC STUDIES (OTHER)

MIPo 82X-RAY EDGE SPECTROSCOPY OF COBALT(I, II, III) B12. Wirt, M., Sagi,I., Chen, E., Frisbie, S., andChance, M., Dept. of Chemistry,Georgetown Univ., Wash., D.C. 20057

We present the first directstructural studies of the cobalt(I,II, III) and "base-off" forms of B12using x-ray edge spectroscopy. Inaddition, model cobalt compounds,including cobalt (I, II, III) di-methylglyoxime (DMG) were examined.A surprisingly small 0.5+.2 eV shiftto lower energy was observed foreach change in oxidation state fromCo*3 to Co+2 to Co+" B12. In general, aone electron reduction of a metalion shifts the edge to lower energyby 1-5 eV. We attribute these smallshifts to a delocalization ofelectron density off the cobalt atomin the two reduced forms. No edgeshift was observed for the Co+3 DMGcompared to Co+2, indicating that theunpaired electron density may belocalized on the axial nitrogen ofthe pyridine ligand. A 2.0 eV shiftto lower energy was observed in thecomparison of Co23 DMG to Co', in-dicating full Co+' character for thatspecies. Also, assignments for othercobalt compounds are presented.M-Po64PURIFICATION OF TRANSCOBALAMIN IIFrisbie, S.M., and Chance, M., Dept.of Chemistry, Georgetown University,Washington, D.C.,20057

Transcobalamin II is responsiblefor transporting vitamin B12 in theblood stream. In order to betterunderstand the mechanisms of bin-ding, we have purified transcobala-min II from chicken serum using anaffinity matrix. Aminopropyl-cobalamin was made from the reactionof cyano-cobalamine with 3-chloro-propylamine, then immobilized onSephacryl (Jacobsen and Huennekens,Methods in Enzymology 123, 1986, 28,with minor changes). The amino-propyl-cobalamin-bound Sephacryl wasused to extract transcobalamin IIfrom the serum. The interaction ofB12 and its binding proteins leads tospectroscopic changes similar tothose observed in B12 dependent en-zyme systems. Therefore, bindingproteins are also a model system forenzyme catalysis. The isolated pro-tein has been used to study protein-ligand interactions using resonanceRaman and other opticalspectroscopies.


M-PosS3FTIR STUDIES OF B12 COMPOUNDS.Taraszka, K., Chen, E., and Chance,M., Dept. of Chemistry, GeorgetownUniv., Washington, D.C. 20057

These studies represent the firs-tIR spectra of B.2 compounds observedin solution and are relevant to B12enzyme dynamics. The major band ofB12 compounds in D20 is observed atabout 1630 cm-' and may be attributedto the acetamide and propionamideside chains of the corrin ring. Thisband is observed at 1633 cm-" forvitamin B12, 1629 cm'1 for coenzymeB12, 1632 cm-' for dicyanocobinamideand is shifted to a higher frequen-cy, 1651 cm-', for "base-off" B12. Inaddition, we have observed a majorband originally seen in solidspectra by Hogencamp et al (J. Biol.Chem., 1965, 240, p.3641) at 1574 cm'for vitamin B12, 1570 cm71 for coen-

zyme B12, and 1582 cm'- for dicyano-cobinamide, while no band wasobserved for base-off B12. This maybe attributed to displacement of Cofrom the plane of the equatorialnitrogen groups. We also report thespectra of Co§2 B12 and aquocobalaminand the observation of furtherspectral modes from 1400-1500 cm-'.

M-PoS5CRYOGENIC OPTICAL SPECTROSCOPY OF B12COMPOUNDS. Chen, E. and Chance, M.,Dept. of Chemistry, GeorgetownUniv., Washington, D.C.,20057

At low-temperature, the vibra-tional splitting of cobalamin pi-piVoptical transitions becomes pro-nounced. Previous investigations(Firth et al.,, Biochem.., 1967, ,

p.2178.) have suggested that a vib-rational overtone of 1300-1350 cnr1exists for many B12 compounds. Wehave re-investigated these spectraat liquid helium temperatures with ahigh resolution intensified diodearray spectrograph. For vitamin B12,we observe the 1,2 progression of1300 cm'i from the secgnd lowestenergy peak. Further towards theblue is a 1,2 progression of 1800 cm-1relative to the second overtone ofthe main band.In adenosylcobalamin,different overtones of 900-950 cm'-are seen. These results suggest thepresence of metal-ligand chargetransfer or d-d transitions inaddition to the classical pi-pitransitions. Since vibrationalmodes are related to excited states,these results are relevant todynamics of cobalamin photolysis.

50a Biophysical Joumal voL 57, 1990

M-Po6..VIBRATIONAL CIRCULAR DICHROISM, RAMAN, FTIRAND ELECTRONIC CD STUDIES OF AZIDE BINDING

TO HEME PROTEINS. S. Ashera, P. Larkina,N. Ragunathanb, T. Freedmanb, L. Nafieb,B. SpringerC, S. Sligarc, R. Nobled.

aDept. of Chem., Univ. Pittsburgh, bDept.of Chem., Syracuse Univ., CDept of Biochem.,Univ. of IL, Urbana, IL, Dept. Med. and

Biochem., State Univ. of NY, Buffalo, NY.

We report novel vibrational CD studiesof azide binding to heme proteins whichwhen coupled to additional optical studiesusing more classical techniques ?clarify?the role of the distal heme pocket indefining the ligand binding geometry andaffinity. We examine sperm whale Mb and a

mutant in which the distal his is replacedby gly, horse Mb, human and carp Hb andproteins reconstituted with modified hemes.

Following the earlier pioneering studyby Marcott et. al. (Optical Activity andChiral Discrimination, Ed. Mason, F.,Reidel Publishing, 1979), the work here isthe first use of VCD to study ligandbinding in heme proteins.

M.PoS8

TI-RESOLVED CIRCULAR DICHROISXSTUDIES OF PROTEINS

Sofie C. Bjorling, Cora M. Einterz,James W. Lewis, Robert A. Goldbeckand David S. Kliger; ChemistryDepartment, University ofCalifornia, Santa Cruz

A technique to measure circulardichroism (CD) spectra with ananosecond time resolution hasrecently been developed. Linearbirefringence and linear dichroismartifacts produced byphotoselection can distort the CDsignal when it is measured duringthe time scale of molecularreorientation. Extensivetheoretical analysis using Jonescalculus has been done to find theconditions where these artifactsare eliminated and the analysis hasbeen tested experimentally. Thetechnique has been applied to thestudy of structural andconformational changes occuring inseveral proteins (including retinaland heme proteins) after theirphotolysis. Theoretical andexperimental results will bepresented.


MPos67SPECTROSCOPIC STUDIES OF THE NUCLEO-TIDE BINDING SITE OF GTPases AND ATPases.John.F. Eccleston, Sally K.A. Woodward, Tazeen F.Kanagasabai and Stephen-R. Martin. National Insti-tute for Medical Research, Mill Hill, London NW7IAA, U.K.

The 2'(3')-O-methylanthraniloyl (mant) deriva-tives of nucleotides are valuable probes of thestructures and mechanisms of GTPases andATPases. Derivatives of ribonucleotides exist as anequilibrium mixture of the 2' and 3' isomers butderivatives of 2'-deoxyribonucleotides consist of asingle isomer. Mant-GDP binds to elongation factorTu (EF-Tu) with the same affinity as GDP and itsfluorescence is enhanced by 60%. Mant-dGDPbinds four-fold weaker than GDP but shows noenhancement of fluorescence. Mant-GDP hasrelatively intense CD in solution which is increasedon binding to EF-Tu whereas mant-dGDP hasweak CD whether in solution or bound to EF-Tu.These results are discussed in relation to the knownthree-dimensional structure of EF-Tu and com-pared with results for the binding of mant-ADPand mant-dADP to myosin subfragment 1 whereboth derivatives show enhancements of fluorescenceand CD on binding.

M-PosS9FUIJR~IE-, EAY (F S-1 OOa E1V1IN.

Len-aw1a2 , Rajam S. ein3, Cyril M.Kay3, axid Herbert C. Chem2. 2Universityof Alabama at Birmingham, Birmingham,Alabama, and 3University of Alberta,Edmonton, Alberta, Canada.

'Ihe emission and anisotropy decays ofthe single tyrptophan of bovine brainS-100a protein was studied by picosecondtime-resolved spectroscopy. With 295 nmexcitation the emission decay was resolvedinto 3 components. At 200C and pH 7.2 thedecay times were 0.43, 1.42, and 4.05ns with fractional intensities of 0.310.31, and 0.38, respectively. Wnile Mg2had a negligible effect on the decaypattern, Ca2 induced a 12% decrease inthe shortest decay time and an increaseof 55% and 14% in the other two decaytimes. The fractional intensity of theshortest decay time was reduced to 0.05,whereas the fractional intensities ofthe other two components were increasedto 0.39 and 0.55. Similar Ca2+-inducedchanges in the decay parameters were ob-served at lower temperatures. The decaykinetics may be indicative of multiplelocal conformations. The anisotropy decaywill be discussed. (Supported by NIH AR-25193 and MRC Protein Group, Canada.)


M-Pos70POLARIZED ELECTIRONIC SPECTRA OF

Z-DNA SINGLE CRYSTALSPui S. Ho, Guangwen Zhon and Leigh B. Clark

Deparanent of Biochemistry and Biophysics, OregonState University, Corvallis, OR 97331 mad Department ofChemistry, University of Califomia-San Diego, La Jolla,CA 92093-0342

Polarzed electronic absopion spectra of the (100) face ofsingle crystals of the Z-form double helical duplex ofd(m5CGUAm5CG) have been oained from Kramers-Kronig analysis of reflection data. The c crystallographicaxis is parallel to the helix axis and shows but weak ab-sorpdon. The b axis is perpendicular to the helix axis andshows a structureless absorpdon band centered at 270 nmwith an oscillator strength of 0.26. These are the first po-larized spectra obtained for a polynucleotide with precise-ly known structure, confonnation and orientation. Calcu-lations of the crystal spectra utilizing available transitonmoment data for the individual chromophores have beencarried through using the oriented gas model (no interbaseinteractions) and, again, employing all base-base interac-tions (point dipole) in the duplex. The calculated hypochromism of the 270 nm band is much less than the exper-imental value obtained from the crystal data, and thisdifference is attributed to chain length effects. The crystalspectra appear to be representative of Z-form double hel-ices of essentially infinite length and not of a collection oftwelve base duplexes. No evidence for nx* transidons po-larized parallel to the helix axis is found.

M-Pos72FLUORESCENCE SPECTROSCOPY OF HUMANIMMUNODEFICIENCY VIRUS-1 revCarlo M. Nalin. Department ofProtein Biochemistry. Hoffmann-LaRoche Inc. Nutley, NJ 07110HIV-1 rev, a viral-encoded proteinessential for replication of thevirus has been purified. HIV-l revcontains a single TRP (residue 45)which is located within an ARG-rich region of the protein (10 ARGwithin residues 35-50). The TRPresidue has an emission maximum at344 nm, characteristic of theamino acid in an aqueous environ-ment. Quenching by acrylamide andiodine was measured and Stern-Volmer constants for each quencherdetermined. Together with polari-zation and anisotropy measurements(0.15 and 0.10, respectively) theresults indicate that TRP-45 islocated on the surface of theprotein. Possible importance ofthe TRP residue and the Arg-richregion in RNA binding will bediscussed.


M-Pos71

THE DETERMINATION OF THE pKa's OFAMINO ACID GROUPS IN PROTEINS BYRAMAN DIFFERENCE SPECTRSCOPY.*Minghe Lee, *Kwok To Yue, +Jie Zheng,+Robert Callender, *Physics Department, EmoryUniversity, Atlanta, GA 30322; + PhysicsDepartnent, City College of CUNY, New York,NY 10031.

Sensitive Raman difference spectroscopy canbe used to study the behavior of small moleculesand/or small molecular groups inside proteins orother large macromolecules (Yue, K. T. et al., J.Raman Spectrosc. (1989) 20, 541-545). Wehave measured the Raman spectra of humantransfernin at different pH's, titrating its varioushistidine residues. For example, the differencespectrum between pH 6 and pH 8 of transferrinis very similar to the difference spectrum ofhistidine in solution, indicating that differemcespectroscopy can be used to study behavior ofindividual residue inside proteins. Similarexperiments with lactate dehydrogenase, whichcontains an essential histidine at its active site,will also be presented.

M-Po873SPECTROSCOPIC EVIDENCE OF HETERO-GENEITY IN THE TRP ENVIRONMENT OFNEUROTOXINSB.D. Schlyer and A.H. Maki, Department ofChemistry, University of California, Davis, CA95616The photo-excited triplet state of the three homo-logous single trp-containing nicotinic acetylcholinereceptor neurotoxins, a-bungarotoxin (BgTX), a-cobratoxin (CbTX) and cobrotoxin (CoTX), havebeen investigated using phosphorescence andoptically detected magnetic resonance (ODMR)spectroscopy. Each shows similar phosphore-scence fiom the functionally invariant trp28 with theintensity ofBgTX and CbTX quenched relative tothat of CoTX. ODMR signals from BgTX andCbTX clearly exhibit multiple trp resonances whichchange features upon varying excitation wavelength.This is intexpreted as heterogeneity in the trp28environment of the unquenched ensemble detectedvia ODMR. CoTX shows no excitation-depend-ence of its ODMR signals. Reduction of the cys29-cys33 disulfide bridge of BgTX (also present inCbTX but absent in CoTX) produces increased trpemission and an ODMR spectrum similar to that ofColX suggesting the role of disulfide in quenchingtrp28. Supported by NIH Grant ES-02662.


M-Pos74

FLUORESCENCE STUDIES OFNONENYMATICALLY GLYCOSYIATEDENZYMES. Aydin 6rstan, Joseh A. Schauerte andAri Gafni. Institute ofGerontology, University ofMichigan, 300 N. Ingalls, Ain Arbor, MI 48109.Nonenzymatic glycosylation ofthe amino groups onenzymes may interfere with their degradation by theubiquitin mediated pathway of protein degradationand result in the accumulation of modified enzymesin cells. As a preliminary evaluation of thishypothesis, we have investigated the susceptibilitiesof several enzymes of the glucose metabolism tononenzymatic glycosylation. When rabbit musclealdolase is incubated with 3 mM fructose- 1,6-diphosphate in Tris buffer, pH 7.5 at 30°C, changesin the absorption spectrum of the enzyme at around330 nm, representing the formation of glycosylationproducts, are observed within 20 hours. Afluorescence emission spectrum (excitation at 360nm), with a peak at 430 nm and a broad shoulder atabout 480 nm, is obtained within less than 5 days ofincubation. The enzyme is also glycosylated byribose-5-phosphate and fructose- I-phosphate.Unphosphorylated glucose, fructose, galactose andribose are, however, ineffective in glycosylating theenzyme even after 37 days of incubation at 30°C.Rabbit muscle glyceraldehyde-3-phosphatedehydrogenase (GPDH) and baker's yeast glucose-6-phosphate dehydrogenase are glycosylated byribose-5-phosphate, producing emission spectraresembling the spectmm ofglycosylated aldolase.

M-Pos76

INTERMOLECULAR INTERACTIONS INMELANIN - A FLUORESCENCE STUDY

C. Pande. Clairl Research Labs., Stamford, CT. 06922.

Melanins are the natural pigments responsible for the color ofhair and sin. They are formed in Wvio by enzymatic oxidationof tyrosine. The main stru nlunits in natual melanins arebelieved tobe 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyin-dole-2-cboxylic acid (DHICA), but the structure is not wellunderstood.

Melanins have unusual optical properties; the absorptionspectra, irespective of the oriin>, are feaurlss, and theabsrxption inaeases monotonously from thene rnfrared to theultravioletregion. Fluescence specrscopy, on theother hand,appears to be quite sensitive to the melanin type1.

Using fluorescence spectroscopy, we show that melanin solu-tions in dimethyl sulfoxide (DMSO) exhibit strong inter-molecular interctions (aggregation). These interactions werenotobserved in aqueous solutions.We attribute these toground-state interaction between the polymeric chains, rather thanexciplex formation, since both the excitation and emissionspectra are affected. For sepia melanin, these interactions resultin ca. 90 nm (4000 cm1) shift in the emission maximum. Thelikely mechanism ofthese intetations and their relevance to theoptical popties of melanin will be discussed.

I C. Pande and T. Schultz,Biophys. J. (abst.) 1989,214a


M-Pos75RESONANCE ENERGY TRANSFER (RET) FROM ACYLINDRICAL ASSEMBLY OF DONORS TO A PLANEOF ACCEPTORS - LOCATION OF THE APO-B100PROTEIN ON THE SURFACE OF THE LDL PARTICLE.J. Eisinger, P. Bastiaens, A. de Beus, H.M.Lacker, P. Somerharju* and M. Vauhkonen*.Dept. of Physiology and Biophysics, MountSinai School of Medicine, New York, NY10029. The rate of RET from a donorcylinder (e.g. a membrane protein) to aplanar acceptor assembly (e.g. membraneprobes) was evaluated by (1) analyticalsolutions for uniform and continuous donor(D) and acceptor (A) distributions, and(2) by computation of the RET rates betweenpairs of discrete D's and A's, for manyrandom distributions of D and A. Resultsfor the two approaches, presented in termsof F6rster distance-independent parameters,are in good agreement, with the latterproviding a measure of the error expectedfor unknown D locations. This methodologyis illustrated by analyzing the RET fromthe 37 tryptophan residues of the apo-B100protein to a series of pyrenyl acceptorprobes in the phospholipid monolayer of thehuman LDL particle and it is concluded thatthe protein is partially immersed in theparticle's phospholipid surface.* Dept. of Medical Chemistry, University ofHelsinki, Helsinki, Finland.

M-Pos77CONSEQUENCES OF THE HYDROGEN BOND

STRENGTH IN THE SYSTEM C=NH+ ....OOC ON THERAMAN EXCITATION PROFILE OF PROTONATEDSCHIFF BASE. IMPLICATIONS FOR BACTERIAL ANDVISUIAL PIGMENTS.

S. ALEX', H. LE THANH1, C. SANDORFY2 ANDD. VOCELLE1. ID6partement de chimie de l'Universite du QuebecI Montr6al, CP 8888 Succ. A. MONTREAL, Qc. H3C 3P8 and2Dpaneent de chimic de llUniversit6 de Montr6al, CP 6128 Succ.A. MONTREAL, Qc, H3C 337 CANADA.

The rctinyl Schiff base (SB. hereafter) was synthesized bycondensation of all-trant-retinal and tert-butylamine and is thechromopbore commonly encountered in bacterial and visual pigmentsunder a protonated form (C-NH+). This SB was then dissolved inchlorofom and complexed with organic acids of pK a ranging fromca.0 to 5 units in water, in order to characterize the consequence ofthe H+ charge perturbation on the x-it* electronic transition of thisdouble bond conjugated system.

The behavior of these compounds was not followed by UVabsorption, but precferably by recording their Raman spectra withvarious excitation wavelengths between 457.9 and 514.5nm, whichare coincident with the long-wavelength side of the X-x* transitionmaximum of the fully protonated SB (This absorption band iscentered at ca.453nm when a strong organic acid is used as a protonsource). All the Raman data collected between 1100-1800 cm1i aredominated by the strong emission of the stretching vibration of theC=C moiety and part of an excitation profile can be drawn from thisparticular mode. Unfortunately, a severe deviation from normalexpectations is observed, due to the presence of species such asC=N....HOOC, C=NH+....-OOC and C=NH+....-OOC....HOOC whichare in cquilibrium and govemed by a multiple-well potential.

In this situation the resonance Reman regime is affected, andcarly theorctical calculations have proved that the appearance of thespectra is more dependent on the displacement of the potentialsurface in the resonating cxcited state than on the population ofeach individual well. In addition, a qualitative pictture of the excitedstate shape can also be deduced. These findings will also be used togive a possible explanation to the color of the retinyl Shiff base invisual pigments.


M-Pos78

CAROTENOID FLUORESCENCE. W.I. Gruszecki,B. Zelent* and R.M. Leblanc. Centre de rechercheen photobiophysique, Universite du Quebec aTrois-Rivieres, Qu6bec, Canada, G9A 5H7.

It has been established that carotenoid pigmentsform aggregates in the hydrated organic solvents[1]. On the other hand, apart from the existingconviction that these pigments do not fluorest,some investigations on the carotenoid fluores-cence have been recently reported [2,3]. We havestudied the fluorescence properties of zeaxanthinand violaxanthin aggregates in water-ethanol (9:1,v/v) solvent system. The analysis of the fluores-cence emission and the fluorescence excitationspectra of these carotenoid aggregates lead us to theconclusion, contrary to the other reports [2,3], thatthe observed fluorescence is related to theradiative deactivation of the symmetrically forbid-den lowest excited electronic singlet state 1A* [4].Along the presented data, the biological impor-tance of the carotenoid fluorescence from the 'A*state will be discussed.1. Hager, A., Planta 29, 38 (1970); 2. Gillbro, T.and Gogdell, R.J., Chem. Phys. Lett. 158. 312 (1989);3. Bondarev, S.L., Bachilo, S.M., Dvornikov, S.S.and Tikhomirov, S.A., J. Photochem. Photobiol.,A:Chemistry 4, 315 (1989); 4. Thrash, R.J., Fang,H.L.-B. and Leboi, G.E., Photochem. Photobiol. 291049 (1979).

M-Pos80BOMBESIN-LIKE PEPTIDE INTERACTIONS WITHMODEL MEMBRANES. P. Cavatorta , G.Farru gia , P. Neyroz , G. Sartor , A.G.Szabo , iDepartment of Physics and 2insti-tute of Biological Chemistry, University ofParma, 43100 Parma, Italy; 3Division ofBiological Sciences, NRC, Ottawa, CanadaKLA oR6.Bombesin (BMB), gastrin releasing peptide(GRP) and litorin (LIT) share a widespreadspectrum of hormonal activity. They act asgrowth factors for certain cell lines. Insolution they exist as an ensemble of un-structured, flexible conformers. With DMPSvesicles an increase of tryptophan fluore-scence and changes in ellipticity show thatthe peptides bind to the lipids with induc-tion of a-helical strulcture. The bindingconstants and amount of induced a-helixvaries in the orde:'Y BMB<LIT<GTRP. The pHbehaviour and salt effects indicate that thebinding is both electrostatic and hydropho-bic in nature. On the contrary, DMPC vesi-cles do not show any detectable interactionwith the peptides. Mixed DMPS-DMPC vesicles,however, show a binding constant similar tothat of DMPS alone. These results suggest aspecial requirement for negatively chargedlipids in the binding process. This require-ment should be important for a membraneassisted hormone-receptor interactionmechanism.

Biophysical Journal vot 57, 1990 53a

M-Pos79

BINDING OF INDOLE DERIVATIVES TOPROTEIN RECEPTORSJ.A. Schauerte, J.S. Chomchai, M.A. Supiano,and A. Gafni* Institute of Gerontology and*Department of Biological Chemistry, Universityof Michigan, Ann Arbor, Michigan. 48109

The mechanisms involved in the recognition ofspeciflc molecules in ligand-receptor complexes involve acomplicated array of interactions. The specific binding ofmany drugs is based upon aromatic ring systems, presumablydue to interactions unique to these residues. Charge transferinteractions, most often involving aromatic components, canprovide high affinity and highly specific binding betweenmolecules. The heterocyclic aromatic molecule indole,commonly involved in charge transfer interactions with othermolecules, is a component of a number of drugs such asyohimbine and serotonin. We are evaluating the bindingaffinity and the specificity of charge-transfer (CI`) complexesthat form between indole-based molecules and proteinreceptors or with electron acceptors, as a function of covalentmodifications on the indole chromophore. These propertiesare being correlated to the ground and excited state electronicproperties of the indole chromophores. hdole derivativeshave been shown to bind to the Alpha-2 receptor of humanplatelets (5-amino indole> indoline> 5-hydroxy indole) aswell as to the enzyme tryptophanase. The binding affimitiesof these derivatives to the protein receptors are not identical,and the properties of these molecules that facilitate interactionwith the receptor systems will be evaluated.

M-Pos8lMYOGLOBIN: EPFFECT OF HEME LIGATION ON THEFLUORESCENCE DECAY KINETICS AND EVIDENCEFOR DISCRETE LIFETIME COMPONENTS. K.J.Willis, A.G. Szabo, M. Zuker, J.M. Ridgewayand B. Alpert*. Div. of Biological Sciences,N.R.C., Ottawa, Canada KlA OR6. *Laboratoirede Biologie Physico-Chimique, Universit6e deParis Vll, 75251, Paris, France.

Myoglobin from sperm whale, purified byHPLC displays picosecond decay kineticsexclusively. Observed lifetimes of 24 psfor Trp 4 and 122 ps for Trp7 (oxy, pH 7)agree with theoretical predictions. Thelifetimes are subtly sensitive to changes inthe heme ligand and pH. The increase in thefluorescence lifetime observed on 02 binding(ca. 6 ps for Trp 4 and 20 ps for Trp7) ismainly due to heme absorption spectrachanges, which reduce the rate of energytransfer from trp to heme. This correlateswith crystal structure data which indicatesthat the trp-heme separation and relativeorientation are unchanged on ligand binding.Analysis of the fluorescence data as a con-tinuous Gamma distribution of exponentialdecays resulted in extremely narrow distri-butions which do not support the existenceof multiple, structurally distinct conforma-tional states in Mb.


M-Po982ROTAMERS AND EXCITED STATE REACTIONS OFTRYPTOPHAN ZWITTERION. A.G. Szabo, K.J.Willis and D.T. Krajcarski. Division ofBiological Sciences, National ResearchCouncil, Ottawa, Canada K1A OR6.

The understanding of the fluorescence pro-perties of tryptophan is vital to its useas an intrinsic probe of protein structureand dynamics. It has been generallyaccepted that at pH 7, the fluorescence oftryptophan decays with double exponentialdecay kinetics, and that this behaviour maybe attributed to different rotamers of thealanyl side chain. The rationalization ofthe increased quantum yield in D20 solutionhas not been completely elucidated. Wehave reexamined the fluorescence decaybehaviour of tryptophan and some of itsderivatives in D20 and H20 under a varietyof excitation conditions and at differentemission wavelengths. The results comple-ment the original'data, but new details ofthe fluorescence decay behaviour, includingexcited state reaction, have been observedwhich may require a reinterpretation of therotamer model. These results will bepresented and possible explanations will bediscussed.

M-PoS84Detection of ATP, ADP, and Phosphate insolution by Near Infra-Red SpectroscopyAndrew L. Mernmelstein, Joan S. Carducci,Richard I. Shrager* and Robert L. BergerLab. of Biophysical Chemistry, NHLBI, NIH

*Lab. of Applied Studies, DCRT, NIHThe direct measurement of ATP, ADP, andphosphate in a buffered solution has beencarried out using a temperature controlled,nitrogen purged cell in an LT IndustriesQuantum 1200 NIR scanning spectrometer.Region scanned was 900 to 1800 nanometersat a resolution of 0.75 nm. Scanning timeis 200 milleseconds. 100 scans were usedfor all concentration ranges from 25 to 1micromolar. All runs were done at 4°C, in0.2M Bis-tris pH 7.40 and 0.1M KC1. At pH8.4, only the phosphate moiety in ATP andADP were seen, thus all samples lookedalike. At pH 6.2, in Bis-tris, ATP and ADPlooked alike but were different fromphosphate. Spectral analysis andcorrelation with concentration is done bysingle-valueddecomposition (SVD) (1,2)and partial-least-squares (PLS) (3) usingMATLAB running on an IBM AT. Extensiveexperience with NIR has been reported inother fields (4,5) but little work has sofar been done in biochemistry andbiophysics. It is hoped this technique maybe of use particularily to workers intransport research.


M-Pos83

RESONANCE RAMAN SPECTRA OF COPPER(II) ANDNICKEL(II) OCTAETHYLISOBACTERIOCHLORIN

David F. Bocian and Alexander D. Procyk

Resonance Raman (RR) spectra arereported for Cu(II) and Ni(II) octaethyl-isobacteriochlorin (OEiBC). The spectrawere obtained by using a variety ofexcitation wavelengths which span the B,Qx, and Qy regions of the absorptionspectrum. The RR intensity enhancementpatterns are found to differ markedly atthe various excitation wavelengths, partic-ularly at different wavelengths within theB band. The majority of the high-frequencyskeletal vibrations of the macrocycle areassigned by examining selectively meso-deuterated CuOEiBC and both high- and low-spin NiOEiBC. Normal coordinate calcula-tions were also performed in order to det-ermine the forms of the vibrational eigen-vectors. Collectively, these studies lendinsight into the structural and electronicproperties of the OEiBC macrocycle.

M-Pos85INFWENCE OF DIFFUSION ON LONG-RANGE ENERGYTRANSFER BY FREQUENCY-DOMAIN FLUOROMETRY.J.R. Lakowlc H. Szmacinskl, 1. Gryczynskl, University ofMaryland Medical School, Department of Biochemistry,Baltiore, MD, and M.L Johnson, University of Virginia,Deament of Pharacology, Charlottesvlle, VA.

We used frequency-domain fluorometry to study long-range intermolecular energy tranfer In presence andabs d transational difusion. Frequency-domain dataof Indole fluorescence (donor) In the absence andpsence of dansylamide (acceptor) In propylene glycol(Pow diffusion) and metnol (high dfusion) were fitted toForster and/or Gosele et al. models, the latter of whichcontains diffusive terms. This donor-acceptor system(Ro - 25A ) is commonly used In intramolecular energytner measurements. The Forster model providesacceptve ffit In propylene glycol, but fals In methanol.The Gosele et al. model gives reasonable fits andParameters (R0 and D) In both solvents. Anaysis dsimulated and measured data show usefulness offrequency-domaIn fluorometry In energy trnsfer studies.From analysis d simulated data we estimated the llmit ofresolution of dfusion coeffiint >D>107cm2/s). In aseparate study, using 2-aminopurine as a donor and2-amlnobenzophSnone as an acceptor, we observedsystematc devWions from the Forster model at higherconcenran dof the accepor. (From the Center forFluorsnce Spectroscopy, University d Maryland.)


MPosS6

DIELECTRIC DISPERSIONS OFBACTERIORHODOPSIN IN THE MHz REGION

Idwar Bakarudin. Institute of BasicSciences, University of AgricultureMalaysia, Bintulu Campus, 97008 Bintulu,Sarawak.

Dielectric measurements around the MHzregion in aqueous solution containingbacteriorhodopsin (bR) have shown theexistance of ,3 -dispersion with fivedistinct parts. ihis dispersion and the

)6 -dispersion at low frequencies conformapproximately to the Debye relaxationequations. The derived apparent relaxationtimes showed dependence on physicalparameters. The relaxation effect at lowfrequencies is attributed to reorientationof bR chromopore within the purplemembrane (PM) fragments. The mechanismwhich give rise to the -dispersion may

well be due to the Maxwell-Wagner effect,although the first two parts of the

Zl-dispersion may also be attributed tocounterion relaxation or bR reorientation.

M-Pos8THE METAL SITE STRUCTURE OF CON

A IN CRYSTAL AND SOLUTIONS-L. Lint, Y. Zhang, E.A. Stern, Dept. of Physics,Univ. of Washington, Seattle, WA 98195, U.S.A.A.J. Kalb (Gilboa), Dept. of Biophysics, The Weiz-mann Institute of Science, Rehovot, Israel.

X-ray Absorption Fine Structure Spectroscopy(XAFS) is employed for an investigation of the com-

parative structure of Concanavalin A (Con A) in formsof crystal and solution. Con A has two distinctive

metal-binding sites. One of these, the transition metal-

binding site, has a different structure and tempera-ture dependence in the two forms. The crystal has

one less ligand than the solution. The crystal also

has an anomalous temperature dependence: The dis-

order of the site is not reduced as the temperatureis decreased, while the metal-ligand bond lengths in-

crease. Normal behavior is observed for the solution

form. We suggest that the constraints of the crys-

tal structure cause a compression in the transition

metal site; the compression increases with tempera-ture, coinpensating the iiormal thermal effect. Our

results suggest caut ,'n in assuming that crystal and

solution forms of proteins are always the same.

[Research supported by NSF grant DMB-8613948]


M-Pos87

CONFORMATIONAL CHANGES OF BOVINEBRAIN SlOOb INDUCED BY TERBIUM ANDCALCIUM. P.L. Pingerelli, M.M. Batenjany andH. Mizukami. Division of Regulatory Biology andBiophysics, Department of Biological Sciences,Wayne State University, Detroit, MI 48202.

SlOOb is an acidic, homodimeric protein whichexposes a hydrophobic domain upon bindingcalcium. Using circular dichroism (CD) and UVdifference spectroscopy, terbium was used to studythe calcium binding sites of SlOOb. The addition ofTb3+ decreased the ellipticity at 222 nm of SlOOb (14gM), and induced a UV difference spectrum, bothsimilar to that observed with calcium. The secondarystructure estimations of the Tb3+ induced far UV CDspectra of SlOOb are consistent with an a-helix tocoil transition, as observed in the presence ofcalcium. The UV difference spectral molar extinctionvalues (A&) at 259.5 nm, characteristic of the blueshifted phenylalanine residues, and at 282 nm,representing the red shifted tyrosine residue, wereless for calcium than for terbium at identicalconcentrations. However, in contrast with Ca2+,higher concentrations of terbium precipitate S lOOb.These data suggest that Tb3+ and Ca2+ have similarbinding sites with a higher affinity for terbium.(supported by a grant from The Graduate School,Wayne State University)

M-Pos89Nonaqueous Hydrogen Bonding and ProtonTransfer by Aromatic Alcohols: Steady-State

and Time-Reslved Fluorescence Studies.C.A. Hasselbacher, L.T. Galati, P.B. Contino,W.R. Laws, 6 J.B.A. Ross Dept. Biochemistry,Mount Sinai School of Medicine, New York, NY

In water, aromatic alcohols, such astyrosine and the estrogen 17fi-dihydroequilenin(170-DHE) which are both fluorescent, canbecome stronger acids in the excited state;proton dissociation may take place on thesame time scale as the fluorescence decay ofthe protonated alcohol. However, the localprotein environment of a tyrosine or anestrogen binding site is not completelyaqueous. To investigate the possibility of thistwo-state excited-state reaction occurring in anonaqueous environment, we have titrated2-naphthol and phenol in organic solvents withthe proton acceptor triethylamine (TEA).Based on shifts in absorption spectra, TEAand the alcohol form a ground-statehydrogen-bonded complex with Ka's on theorder of 100 M-1. From shifts in steady-stateemission spectra and the characteristics of thefluorescence intensity decay kinetics, we findthat any subsequent excited-state protondissociation depends on solvent polarizability.Furthermore, alcohol not complexed with TEAis dynamically quenched by TEA.Supported by NIH grants GM39750 and DK39548.


M.PoSO9Tyrosine Fluorescence in Oxytocin: Rotamers

and Neaest Neighbor Interactions.Paud B. Contino and William R. LawL

Department of BiochemistryMount Sinai School of Medicine, New York, NY

Oxytocin (OT) is a nonapeptide hormonecontaining a single aromatic residue, Tyr2.The Cyst * CysS disulfide bond staticallyquenches one of the three Ca-Ca phenol sidechain rotamers [Biochemistry 25, 607 (1986)].We now report that the N-terminal aminogroup also influences the phenol environment.teady-state Stern-Volmer fluorescence

quenching studies (pH 3) indicate that theapparent quenching constant, k foracrylamide is smaller for tyrosine in thanfor two desamino OT analogs, suggesting sterichindrance by the amine. Furthermore, theapparent k of iodide ion is larger for OTthan for the analogs, indicating electronicattraction by the protonated N-terminalamine. The disulfide bridge does not,however, significantly affect the efficiency ofthese fluorescence quenching agents. Initialtime-dependent fluorescence quenching studiesindicate that each Ca-C6 rotamer has adistinct k for each quenching agent,supporting t9he concept, based on their distinctlifetimes (ibid.), that each rotamer has adifferent environment. Supported by NIH grantsDK39548, DK10080, and GM39750.

M-Pos92

Trptophan-A1 and Tryptophan-As Insmlins:Steady-State and Time-Resolved Fluorescene.

N. Ohta, L. Zong, P.G. Katsoyannis,W.R. Laws, (4 J.B.A. Ross Dept. Biochemistry,Mount Sinai School of Medicine, New York, NY

The A and B chains of insulin eachcontain two tyrosine residues. To study thelocal environments of the A14 and AID tyrosinesites, and to study the fluorescencecharacteristics of a tryptophan residue in awell-defined system, a tryptophan residue wassubstituted for either tyrosine-A'4 or AiD.Usually, chemical modification of tyrosine-A14has little effect upon biological activity, butmodification of tyrosine-A19 generally resultsin diminished activity; the same behavior isobserved for the tryptophan analogues. Thefluorescence emission of tryptophan-A14 iscomparable to that of model compounds, butthe emission of tryptophan-Al has its peak4 nm to higher energy. The fluorescenceintensity decay of each tryptophan analogue iscomplex but emission wavelength independent.WVhile each tryptophan has similar kineticconstants, the mean intensity decay times ofthe A14 and A19 analogues are 5.7 ns and3.3 ns, respectively, since each similar kineticconstant has a different fractional intensity.The fluorescence results can be correlated withthe local environments of the probe residues.Supported by NIH grants GM39750 and DK12925.


M-PoS1

[Lys(DNS)' Vasotocin: A Eighly FluorescentNeurohypophyseal Hormone Analog.C.A. Hasselbacher, G.P. Schwartz,

6 W.R. Laws, Department of Biochemistry,Mount Sinai School of Medicine, New York, NY

and J.D. Glass, Medical Department,Brookhaven National Laboratory, Upton, NYThe neurohypophyseal hormones oxytocin

(OT; lactation and uterine contraction andvasopressin (VP; renal water retention) arehomologous nonapeptides cycized by a Cyst +

CysS disulfide bond. These hormones bind totheir storage/carrier protein neurophysin (NP).To measure binding interactions, we havesynthesized a dansyl (DNS) OT analog,[Lys(DNS)B] vasotocin, by conjugation of thecyclic tocinoic acid (-S-Cys-Tyr-Ile-Gln-Asn-Cys-S) with the "vaso" tripeptide Pro-Lys(DNS)-GlyNH2. In aqueous solution(100 mM KCl, pH 6.4 to 7), the DNS groupof the OT analog displays fluorescenceproperties characteristic of fully-hydrated DNSmodels. This OT analog is capable of tight,reversible binding to NP, as shown by anincrease in fluorescence intensity, a corres-ponding spectral shift of the emission to higherenergies, and an increase in fluorescenceanisotropy for the DNS probe on titrationwith NP; these changes are lost upon additionof an excess of native OT. Supported by NIHgrants DK39548 and DK10080.

M-Pos93FLUORESCENCE OF' TRYPTOPHANDIPEPTIDES. R.F. Chen and J.R.Knutson, Lab. of Cellular Biology,National Heart, Lung, and BloodInstitute, National Institutes ofHealth, Bethesda, Md. 20892.

When comparing dipeptides of thetype W-X and X-W, where W = Trpand X = another amino acid, thefollowing were found for W-X :1. Absorption and emission spectrawere blue shifted. 2. Quantumyields were higher for bothzwitterionic and anionic forms.3. pK, values for the amino groupwere lower. 4. The fluorescencedecays were more nearly exponen-tial. The parameters for W-W werelike those for X-W, showing thatthe C-terminal W acts as an energysink. These results, as well as thedecay-associated spectra, suggestpredominance of a rotamer of W-Xwhere the indole and -NH3+ groupsare in close proximity. Whilespectral shifts in proteins areusually interpreted as reflectingdegree of W exposure, the presentresults indicate that electrostaticfactors can play a part.


M-Pos94

DISTRIBUTION OFTOMAYMYCIN BONDED TODNA. Mary D. Barkley, Fahmida N. Chowdhuryand Karol Maskos, Dept. of Chemisty, LouisianaState University, Baton Rouge, LA 70803

The fluorescence decay of tomaymycin bonded tocalf thymus DNA is biexponential with lifetimes of3.5 and 6.7 ns at 50C. Studies with synthetic DNAoligomers and polymers show that the values of thelifetimes and the relative amplitudes of the twoexponential components depend on the flanldng basesequence of the covalently modified guanine.Tomaymycin and other pyrrolo[1,4]benzodiazepineantibiotics have four possible bonding modes: twostereochemistries of the covalent linkage and twopossible orientations of the drug on the DNA helix.The two fluorescence lifetimes of tomaymycinprobably represent the two stereochemical linkages.To determine whether the two exponential decaysrepresent particular adducts at preferred bondingsequences or a distribution of adducts, thefluorescence decay data were fitted to discrete andcontinuous lifetime distributions. Time-resolvedfluorescence data firom experiments performed atdifferent excitation and emission wavelengths, pH,DNA concentration and chain length, andnucleotide/drug ratio were analyzed simultaneously inglobal programs. Supported by NIH grantGM35009.

M-Pos96REACTIONS OF EXCITED TRIPLET STATES OF PdAND Zn MYOGLOBINS C. M. Phillips, J. M. Vanderkooi& S. Papp, Dept. of Biochem. & Biophys. and Dept. of Chem.,Regional Laser & Biotechnology Laboratory, Univ. ofPennsylvania, Philadelphia PA 19104The triplet state ofZn and Pd derivatives of myoglobin werestudied by transient absorption and emission techniques. Thetransient absorption spectra following laser flash excitationshowed a new absorption band at 460 nm and 418 nm for theZn and Pd derivatives, respectively, with decay parameterscomparable to phosphorescence. For Zn-myoglobin, thespectra did not exhibit an isobestic point and the decaykinetics to the ground state depended upon the wavelength ofmeasurement, indicating that there was more than one formof emitting species. For Pd myoglobin, a single exponentialdecay and an isobestic point on the transient absorptionspectra were observed. Possible reason for these differentdecay phenomena can be related to the different geometries:Zn porphyrin, five coordinated, can undergo out-of-planedistortions (as does deoxy-myoglobin) whereas Pd, whichcannot accept a fifth ligand, is likely to form a derivative whichresembles oxj myoglobin. jlhe puenching rate constant foroxygen at 20 C was 9.7x10 M s for both derivatives,suggesting no difference in oxygen penetrability. Thequenching by anthraquinone sulfonate was examined on atime scale of psec to msec. The data suggest that a fasttransfer process occurs, without indication of a formation of atransient cation. This result would be predicted forporphyrins in hydrophobic environments as in the hemepocket. (Supported by NIH Research Grants GM 21487 and5-P41-RR-01348-08).


M-Po0S5

TRYPTOPHAN EXCITON COUPLING IN THE fd PHAGEGregory E. Arnold & A. Keith Dunker, Washington StateUniversity, Pullman, Washington.

The fd phage coat protein (8P) contains 50 amino acids,and is predominantly helical. The bulk of the fd capsidconsists of a tight interlocking network of about 2700copies of the 8P protein arranged as closely packed helicallayers, with their axes approximately parallel to the particleaxis. The CD spectrum of the native virus has a highlyunusual shape, exhibiting both the 208 umn -x* and 222un n-x* transitions typical of a helix, but with the 222un trough being significantly deeper than the one at 208un. Previous interpretations ascribed this irregularity tooptical distortions (absorbtive flattening and/or differentiallight scattering) arising from the large particulate size ofthe virus as compared to a protein uniformly disbursed insolution. It is our contention that the anomalous CDintensities arise, not from optical artifacts, but rather from8P tryptophan absorbtion. We present the results of CD,Raman, UV, and fluorescence studies on the oxidation ofthe fd virus by NBS. These studies suggest that NBS, inlow concentrations, specifically oxidizes the 8P tryptophanwithout altering the secondary structure of 8P and,concomitantly, eliminates the anomalous spectral shape. ACD difference spectrum (fd spectrum - NBS treated fdspectrum) provides evidence that the unusual spectral shapeis, in large part, due to exciton coupling betweentryptophans in the 8P capsid network.

M-Pos97

PROTEIN PHOSPHORESCENCE QUENCHINGBY DIFFUSION ENHANCED ENERGY TRANS-FER J.V. Mersol, A. Gafni, M. Model, D.G. Steel(Intro. by Robert Zand) The University ofMichiganAnn Arbor, MI 48109.Room temperature tryptophan phosphorescence fromdeoxygenated aqueous protein solutions can be usedfor detailed study of protein stmcture and dynamics.In this study the distances of closest approach betweenphosphorescent tryptophan residues in several pro-teins and freely diffusing quenchers of various sizeswere determined, using resonance energy transfermeasurements in the rapid diffusion limit. From therate of quenching of alkaline phosphatase (AP) phos-phorescence by the heme group ofmyoglobin (Mb)due to dipole-dipole energy transfer, a distance ofclosest approach of 21 A between this group and thephosphorescent tryptophan was obtained, yielding adistance of 16 A from the latter residue to the surfaceofAP accessible to Mb. In contrast, using freely dif-fusing small dye molecules as energy acceptorsyielded distances of "'2 A for the depth of the sameresidue beneath the enzyme surface. This apparentdiscrepancy is alleviated by assigning the phosphores-cence to Trp 109 in AP which is close to the surface ofthe active site cleft into which the small dyes, but notMb, can diffuse. The unrealistically small distances ofclosest approach obtained with the dyes also reflect thefact that mechanisms other than resonance transferdominate the quenching rate below -10 A. These in-clude electron transfer and exchange interactions.


M-PosOSANALYSIS OF THE SELF ASSOCIATION OF BIIRUB-IN DITAURATE (BDT) AND THE INFLUENCE OFBILE SALTS USINGANALYTICAL ULTRACENTRIFUG-ATION. 'Ridgeway, T.M., 2Carey, M.C., 3Neubrand, M.W.and 1Laue, T.M., 'Dept. Biochemistry, U. New Hampshire,Durham, NH 03824, Harvard Med. School, Boston, MA02115, 3Klinikum Gro#hadern, Munich, Fed. Rep. Ger-manyBilirubin ditaurate (BDT, Mr=850) is a good analog tothe unstable bilirubin glucuronide conjugates that con-stitute the major bile pigment of humans. It is ofphysiological importance to understand the secretion ofbilirubin conjugates. Therefore, the aggregation statemust be assessed under a variety of conditions, includingthe effects of various bile salts. The extent of BDTassociation has been examined as a function of BDTconcentration at fixed ionic strength, as a function ofionic strength at fixed BDT concentration, and in thepresence of different taurine-conjugated bile salts at non-micellar concentrations. BDT self associates to tetramer,with association facilitated by increased ionic strength.BDT self association and BDT association with bile saltappear to result in spectrally distinguishable complexes.Advantage may be made of the spectral differences todetermine the effects of BDT association with bile saltson BDT self association. Supported by NSF BBS 86-15815.

M-Posloo

A CONFORMATIONAL STUDY OFSYNTHETIC ANALOGUES OF THE

SIDEROPHORE ENTEROBACTIN USINGVIBRATIONAL CIRCULAR DICHROISM

-M. Germana Paterlini, T. B. Fredman, L. A. Nafie,Department of Chemistry, Syracuse University,Syracuse, NY 13244; Y. Tor, A. Shanzer,Department of Organic Chemistry, Weizmanninstitute of Science, Rehovot 76100, Israel.

We have investigated the solution con-frmatons of three trpodal peptides, R(CH NH-COCHRRNHCOBoc)3, withR = Ph or N(Ch2)3 andR'= CH3 or CH2C H th), tat serve as precursorsin the synthesis of iron b ners. Interchain hydrogenbonds between the amino acid carbonyl of one chainandNH of an adjacent chain can form, resulting in aconformer wit C3 symmetry. From the vibrationalcircular dichroism (VCD) spectra, we were able todeutmine th handedness and extent of the hydrogenbonding networ. The compounds were studied inCHC13 or C2C14 solution at a concentration of5 x 1O8 M. Spectra were recorded in the NH andcarbonyl stretching regions. The (+,-) VCD coupletobserved at 1676 cm arsing from the amino acidcarbonyl stretches is consistent with a clockwisearrangement of the amino acid carbonyl groupsabout the C3 symmetry axis. Interchain hydrogenbonded NH stretches give rise to a negative VCDband at 3350 cm7l, which correlates with theorientation of the amino acidNH bonds.


M-PoO99CONFORMATIONAL ANALYSIS OF 8-CASOMORPHIN ANALOGUES BYSPECTROSCOPIC AND BIOACTIVITYMEASUREMENTSD.E. Eppsl, H.A. Havell, N. N. Chungl, P.W.Schiller2, T.K. Sawyer2, D.J. Staples2, B.Hartrodt3 and A. Barth3The Upjohn Company, Kalamazoo, Mll,Clinical Research Institute of Montreal,Montreal, Quebec, Canada2, Martin LutherUniversity, Halle, DDR3

The solution conformations of a series ofTrp3 substituted B-casomorphin-5 analogswere investigated by spectroscopic andbioactivity (BA) measurements. Sequenceswere identical with variance only at Pro2 andPro4 positions. CD measurements revealed adirect correlation between certain CDparameters and BA activity. Fluorescence (F)energy transfer experiments established arank order correlation between the Tyrl-Trp3intramolecular distance and BA. F (T) data ofall analogs were best fitted to a doubleexponential decay model. Short-t valueswere shown, by global analysis of the decaysto be identical for all three analogs studied.The results of our biological andspectroscopic experiments indicate thatcasomorphins which can form pseudocyclicstructures in solution are preferred forbinding to Vi receptors.M-Pos101IONIC SPECIES IN HEMATOPORPHYRIN IX IDEN-TIFIED BY FLUORESCENCE SPECTROSCOPY, C.Chapados, D. Girard, and M. Ringuet, Depar-tement de chimie-biologie, Universite duQuebec a Trois-Rivieres, Trois-Rivieres, QCG9A 5H7; P. Nadeau, R. Pottier and G. Wea-gle, Department of Chemistry and ChemicalEngineering, Royal Military College of Ca-nada, Kingston, ON, K7K 5LO, Canada.

The protonation state of the imino nitro-gen of hematoporphyrin IX (Hp) in solutionin water or in a mixture of SDS in wateris evaluated by fluorescence spectroscopybetween pH 0.1 and 13. At pH greater than6 the prodominant species is the free baseand at pH lower than 2, the predominantone is the dication. In water solutionsthe spectra are complicated by aggregation.In SDS solutions, the problem is less se-vere. The spectrum of the free base andthat of the dication are subtrated fromthe spectra of the species obtained at in-termediate pH to obtain the spectrum ofthe monocatton. The relative amount ofthe dication, monocation and free basealong with the aggregation formation of Hpas a function of pH are discussed.


M-Po 102TIME-RESOLVED FLUORESCENCE USEDTO ANALYZE pH-DEPENDENT CHANGESIN THE UNFOLDING KINETICS OF IL-1 . P. HENSLEY+, P. YOUNG+, T.PORTER+, D. PORTER-, K. KASYAN+AND J. KNUTSON-. + Smith, Kline,and French, King of Prussia, PA.,19406,- LC, NHLBI, NIH, Bethesda,MD, 20892.

Interleukin-I6 ,an importantimmunomodulator, is a single Trp,17kD protein, displaying a pH-dependent fluorescence intensityprofile (pKa#v6.6). GdmHCIunfolding kinetics are alsomoderated by pH. We have employeda laser driven, time-resolvedfluorescence spectrophotometerto: 1. obtain decay associatedspectra (DAS) of wild type andmutant proteins vs. pH, 2. obtainanisotropy profiles andcorrelation times for each form,and 3. obtain decay profilesevery few seconds duringfolding/unfolding reactions("KINDK"). The loss of a native

,5.3 ns decay component andproduction of a-1.8 ns termdominates these kinetic profiles.

M-Posl 04

ANALAZING THE DECAY CONSTANTS IN PHASEMODULATION MULTIFREQUENCY FLUOROMETRYUSING THE 11RXll1UII ENTROPY JIETHOD (liEM).R.K. LIIUESEY+,J.C. BROCHOI *,J. POUGETxB. URLEURx / +: MlRC-DRllTP Cambridge U.K.*: LURE Orsay France,x: CHRII ParisMEl is used to recover the distribution

of exponentials describing the fluores-cence decay without any prior model.Thechi-squared function is used to bounda solution. MEn can handle sharp,broad,and continous distributions of lifetimesThe programm was tested by analyzingsynthetic data with different magnetudeof statistical error added.The widths ofrecovered distributions were related tothe noise level,the frequency domain usedand the number of data.The resolvabilityof closed distributions was studied fromsimulated data.fEf was applied to realdata in analyzi'ng e'ither phase and modu-lation data simultaneously or phase andmodulation data separately in order totest the influence of both statisticaland systematic errors.

Biophysical Journal vol 57, 1990 59a

M Pos103QUANTUM AND MOLECULAR MECHANICAL STUDIESOF 2-PYRIMIDINONES AS FLUORESCENT PROBESOF NUCLEIC ACID STRUCTURE. J.D.Petke,P.H.Lee, G.M.Maggiora, D.C.Rohrer, andR.E.Christoffersen, The Upjohn Co.,Kalamazoo, MI 49001

The electronic spectrum of 2-pyrimidinonewas computed using ab initio configurationinteraction methods with an extended basisset. The calculations showed that thelowest spectral band contains a singleintense transition at 310-315 nm. Furthercalculations on 5-amino-2-pyrimidinoneshowed a red shift of the band to 340 nmwith no loss of intensity. Thus,2-pyrimidinones with electron-donatingsubstituents at the 5-position appear tobe potential sources of fluorescense withina window of the nucleic acid spectrum, andit is proposed that such moleculesselectively substituted for cytosine inpolynucleotides may be used asfluorescent structural probes. Molecularmechanics studies of double-stranded DNAdodecamers in which one cytosine wasreplaced by 5-substituted 2-pyrimidinonesin each strand showed little distortion ofthe helix, even though a hydrogen bondingsite is lost.

M Pos1 05SURFACE EXCITED SOLID STATE

LUMINESCENCE SPECTRA OF LANTHANIDE-ATP-BISPYRIDYLAMINE COMPLEXES:

VIBRONIC FEATURES

William R Kirk, Max Plank Inst.f. Biophys. Chem, D-3400 G5ttingen

The success of Cini et. al.(J.Chem.. Soc. Dalton Trans. 1984,2467)in obtaining a stable solid complexof ATP with bispyridylamine and di--valent metal cations was exploitedslight modifications to obtain thefollowing complexes: Tb(III):ATP:bipy , Eu(III):ATP:bipy ,Eu(II):ATP:bipy ; whose spectral featureswere compared with those of Gd:P,.07and Gd:P0 CH.. PO.3 , especially inthe region of vibronic "satellite"transitions (cf. Timofeev et al.Opt. Spek. 1984, 56(3), 250, Stavolaet al. J. Chem Phys. 74(8), 4228,1981). These studies may help indetermining such vital informationas the change in force-constant ma---trix of P-0, O-P-0, P-0-P, etc.vibrations upon metal complexationin biologically relevant polyphos--phate systems.


M-PoslOSDYNAMICAL TEST OF DAVYDOV-TYPE SOLITONIN ACETANILIDE USING A PICOSECOND FREE-ELECTRON LASERMark Roberson, Robert Austin: Princeton UniLewis Rothberg: Bell LabsWunshain Fann; Stanford Univ.John Madey, Steve Benson: Duke Univ.Shahab Etemad: Bell Core

Picosecond Infrared excitation experimentson acetanilide, an alpha-helix protein ana-log, indicate that the anomalous 1650 cm'1band which appears on cooling of acetanilidecrystals persists for at least 2 microseconds following rapid pulsed heating. Theground state recovery time is 15 picosecondsconsistent with a conventional vibrationalmode strongly coupled to the phonon bath.We therefore suggest that the unusual tem'perature dependent spectroscopy of ACN canbe accounted for by a slightly non-degen-erate hydrogen atom configurations in thecrystal and not an unusual localizedvibrational state.


M-Posl 07

COLLAGEN MONOMERS UNDERGOACONFORMATIONAL TRANSITION PRIORTO ASSEMBLY TO FIBRILS. A. George andA. Veis. Northwestern University, Chicago, IL60611 The assembly of collagen molecules intonative fibrils can be accomplished in vitro insolutions at low ionic strength and neutral pH byraising the temperature above 300 C.Precipitation is not immediate but exhibits adistinct lag period. One proposal suggests thatthe lag phase is evidence for a conformationaltransition in the collagen monomerprior toassembly. Fourier Transform Infra RedSpectroscopy (FTIRS) is a very sensitive probe ofthe H-bonded states within the collagen tripleheli.. The carbonyl group spectrum (1700 to 1600cm ) has been investigated in collagen H20solutions at 1 mg/ml under self assemblyconditions from 40 to 340 C. The tIree clearbands at 1660, 1642 and 1630 cm vary mirelative intensity over this range. The 1660 cm'band, associated with the triple helixconformation, increases in intensity at260 relative to both lower and higher T. Fromthese data we hypothesize that the the triple-helixof the semiflexible collagen molecule is actuallyperfected during the lag phase, facilitatingnucleation and intermolecular interaction, butthat the helix must be distorted as the moleculesare bent as they are incorporated into the fibrils.Supported by NIH- NIAMSD Grant AR-13921.

NUCLEIC ACID-PROTEIN INTERACTIONS, INCLUDING CHROMATIN

M-PoslOS

A ZINC BINDING PEPTIDE BINDS TONUCLEIC ACIDS. Martha Delahunty, RichardKarpel and Michael Summers. Intro. byMarIa Frelre. Department of Chemistryand Biochemistry, UMBC; Baltimore, MD.

A fluorimetric method was used to studythe Interaction of a zinc-binding peptidewith nucleic acids. The synthetic peptidecorresponds to the first of two putativezinc-binding domains of the HIV nucleo-capsid protein p7. The .zinc binding domainIs completely conserved In retroviral nuc-leocapsid proteins as well as In eukaryotictransposable elements. This sequence ho-mology may reflect a shared requirementfor Interactions with nucleic acids. The 18aa peptide used In these experiments hasbeen shown by NMR methods to bind zinctightly and stoichlometrically. Here, weexmine the effect of zinc on the Interactionof this peptide with nucleic acids by fol-lowing the fluorescence enhancement ofpolyethenoadenylic acid (poly(EA)) uponbinding peptide. Zinc-bound peptide has agreter affinity for poly(EA) than does thepeptide alone. By competition experiments,we find that both single-stranded anddouble-stranded DNA compete for peptidebinding with poly(C-A).

M-Posl 10DNA FOOTPRINTING AT HIGH PRESSURE

RobertB Macgregor Jr, Carollyn S Ogksby &Benny CAslcew

Molecular Biophysics Research DeparmentAT&T Bell Laboratories

By the use of photoactive DNA cuttingreagents, the interaction between DNA andproteins can be investigated at the singlebase pair level at elevated pressures. Theperturbation induced by changing thepressure reveals regions of the protein-DNAcomplex that differ in their relativeinteraction strengths, thus providing ameasure of the micro-association constants.High pressure footprinting data of therestriction endonuclease E co RI with itsrecognition site will be presented andcompared with thermodynamic andstructural data for the DNA complex. Adescription of the technique and adiscussion of its potential for investigatingprotein-DNA interactions will also bepresented.

M-Posl09A NEW DNA BINDING MODE FOR CAP

J. Michael Hudson and Michael G. FriedThe University of Texas Health Science Center

San Antonio, Texas 78284-7760

In the absence of cyclic AMP, the Ehichiacoli cyclic AMP receptor protein (CAP) binds withoutdetectable sequence specificity to restriction frag-ments containing Ja and a promoter sequences.Under standard conditions (10mM Tris, 1mM EDTA,pH 8.0), our estimates of the equilibrium constant andcooperativity parameter for complex formation are114,000 ± 1400 M-1 and 1.3 ± 0.8, respectively.Thus, this interaction lacks the substantial coop-erativity previously reported for CAP binding to geno-mic DNAs. Using the electrophoresis mobility shiftassay we find that complexes of increasing CAPcontent differ by a highly uniform mobility decrement.This result is most consistent with a binding mode inwhich little or no DNA bending occurs. The ability ofCAP to distinguish between restriction fragments andgenomic DNA, shown by the difference in binding co-operativity, suggests the existence of previouslyunsuspected DNA sequences or structures thatmodulate its binding cooperativity.

M-PosolIVISUALIZATION OF T4 REPLICATION ACCESSORY

PROTEIN COMPLEXES ON DNA BYCRYO-ELECTRON MICROSCOPY

Ed Gogol, Mark Young, Bill Kubasek,Thale Jarvis and Pete von HippelInstitute of Molecular Biology

University of Oregon, Eugene, OR 97403

DNA replication in bacteriophage T4 isperformed by proteins encoded in the T4genome, which act in a multisubunitassembly. While the gene 43 product con-tains the essential polymerase activity,its function is dramatically enhanced by aset of "accessory" proteins, the gene 44,62, 45, and 32 products. Together, theyraise the polymerization rate, fidelityand processivity of the polymerase complexin an ATP-dependent manner, to levelswhich approximate those in vivo.

We have examined complexes formed bythe accessory proteins bound to DNA bycryo-electron microscopy, in the absenceof stains or other potential perturbants.Distinctive structures, approximately 90Aby 30A, are clearly visible crossing theDNA strands at right angles. Theirassembly and maintenance requires all fouraccessory proteins and ATP. Thecomposition of these complexes and thearrangement of their components iscurrently under investigation.

Biophysical Joumal voL 57, 1990 61a


M-Posl12

ROLE OF POLYAMINES IN THE INTERACTION OF GENE-REGULATORY PROTEINS WITH DNA: ESTROGEN ANDPROGESTERONE RECEPTORS. Thresia Thomas andT.J. Thomas, Departments of Env. and CommunityMedicine and Medicine, UMDNJ-Robert WoodJohnson Medical School, Piscataway, NJ 08854.

Hormonal regulation of gene expression involvesthe interaction of the hormone to its specificreceptor protein and the recognition of the receptorby the hormone-response elements in the genome. Togain insight into the mechanism of receptor-DNAinteractions, we studied the binding of estrogen (ER)and progesterone (PR) receptors to poly(dA-dC).-poly(dG-dT) in the presence of a natural polyamine,spermidine3+. ER and PR allowed to bind to DNA-cellulose and then eluted with the polynucleotide. Inthe absence of spermidine, 1400 and 250 rg/mI ofthe polynucleotide was required to elute 50% of ERand PR, respectively from DNA cellulose. In thepresence of 150 gM spermidine, only 65 and 50jg/mI of the polynucleotide was required to elute 50%of ER and PR. We also found that the polynucleotideassumed a left-handed Z-DNA-like conformation inthe presence of 150 jM spermidine. Controlexperiments poly(dA-dT).poly(dA-dT) that does notassume the Z conformation showed no enhancedbinding of ER and PR in the presence of spermidine.Our results suggest that polyamine-inducedconformational transitions may be a part of themechanism of hormonal regulation of gene expression.M-Posl 14

CALCULATION OF MACROMOLECULAR CROWDINGEFFECTS ON PROTEIN-DNA INTERACTIONS IN VIVOMARK M. GARNER, D. SCOTT CAYLEY, AND M.THOMAS RECORD, JR. DEPTS. OF CHEMISTRY ANDBIOCHEMISTRY, UNIVERSITY OF WISCONSINMADISON, WI 53706.

In in vitro assays protein-nucleic acid interactions areexquisitely sensitive to salt concentration changes. ForE. coli grown in minimal medium, increased extracellularosmolarity results in a four-fold increase in cytoplasmic[K+]. This increase has no effect on the extent of lacrepressor occupancy of the lac operator or on theinitiation rate of RNA polymerase at the XPR promoter,even though such a change would abolish theseinteractions in vitro . In addition to increasing [K+]cyt,high external osmolarity results in a 56% increase incytoplasmic protein concentration. Calculations ofthermodynamic non-ideality due to excluded volumeeffects using scaled particle theory, assuming areasonable distribution of sizes for cellular proteins andsupramolecular assemblies, show that increases inprotein activity coefficient due to increasedmacromolecular crowding could offset the decrease inaffinity constant due to the increase in saltconcentration. Hence, the extent of DNA bindingwould not be expected to decrease. We argue that thisincrease in excluded volume non-ideality is one of thephysical chemical principles which allow the cell tomaintain functional homeostasis in the face of changingcellular composition.

M-Poul13

DNA-BINDING CHARACTERISTICS ANDTHERMAL STABILITIES OF NATIVE ANDMUTANT CRO REPRESSORS. James D. Balejaand Brian D. Sykes, Department of Biochemistry,Univ. of Alberta, Edmonton, AB, Canada T6G 2H7.

Cro repressor is a small dimeric DNA-bindingprotein from bacteriophage lambda. The X-raystructure of cro shows that a-helices of a helix-turn-helix motif of each monomer can interact with baseswithin successive major grooves of DNA. In thecomplex, either the DNA or the protein must bend inorder to achieve a better topological fit and to explainchemical modification data. In the cro repressor,valine 55 of one monomer is near enough to valine55 of the other monomer that, upon replacement ofthis residue by cysteine, an intersubunit cross-linkforms without significantly perturbing the structure.Circular dichroism and IH-NMR spectra show thatthe folding of the covalent dimer of the variant(V55C) is nearly identical to that of the non-covalentdimer of wild-type cro. However, individualbackbone amide proton exchange rates and thermalstability measurements indicate that V55C is lessflexible. Upon protein-binding, chemical shiftchanges are induced for the base-pair imino protonsof DNA which reflect binding stoichiometry andstrength. The variant binds about 8 times moreweakly than the native protein. Adjustments inprotein structure, necessary to form a tight protein-DNA complex, are hindered by a loss in proteinflexibility caused by the intersubunit cross-link.

M-Pos 15

s5oiW-mun PE'IIIEw BOWFC3T3 EORTIIN CO LIF CXM)ITIOES. Kim & D. Bickar. Dept. of Chmistry,Smith College, Northapon, MA 01063

Peptide bornd formation occursspontar~ously in solutions containingiron(II), alanine, arxn the rmcleotidebase adenine. We found peptide fotionto require all three components, and tobe further enhaned by the addition ofuracil. Based on the affinity ofiron(II) for amino acids and rncleotidebases, and fro information about thebase-stacking characteristics ofrnucleotide bases in solution, we composeda model for a nucleotide base-iron(II)-amino acid complex which explains theformation of peptide bonds in solutionscontainirx these coiponents. The modelis consistent with nonar 1o polypeptideforMation and informtion transfer, andmay provide the missing link in thesequce from single component moleculesto self-replicating fonri.



M-PoslI6

PEPTIDE BINDING TO POLY ( dG-dC), AVIBRATIONAL CIRCULAR DICHROISMSTUDY.M.S. Gulotta, M. Diem' and D.J.Goss, Hunter College of CUNY, NY.

Many techniques have been usedto examine peptide binding to DNA.Vibrational (ir) circular dichroismhas already been used to describepeptide conformations as well asthe helical structure of variouspolynucleotides and of DNA.Distinct spectral differences areseen for B and Z DNA. We havestudied the effects of peptidebinding to poly(dG-dC) using VCDand compared these effects to thoseseen in high salt solutions ofpoly(dG-dC). The spectra suggestthat the helix remains in a B-conformation, however the positivepeak of the bisignat couplet in thecarbonyl region is greatly enhancedrelative to the negative peak. Weare currently developing atheoretical model to interpretthese results. Grant Support: AHA,NSF86007070, NIH28619GM.

M-Posol8

SEQUENCE-SELECTIVE BINDING OF DNA TO THEREGULATORY SUBUNIT OF cANP-DEPENDENT PRO-

TEIN KINASE. Joe C. Wu, Johnson Lin andJui H. Wang, Bioenergetics Laboratory,Acheson Hall, SUNY, Buffalo, NY 14214.

The regulatory subunit (RII) of type IIcAMP-dependent protein kinase binds thepalindromic octanucleotide d(TGACGTCA) withconcomitant decrease in the fluorescence ofits Trp-226. Scatchard plots of the fluoro-metric data show that each subunit bindsonly 1 octanucleotide with Kd 80 nm and2 'M for RII(AMP)2 and RII respectively.The Kd values of RII(cAMP)2 for non-speci-fic octanucleotides and thymus DNA are 1 to2 orders of magnitude higher. The complexformed by RII(cAM2)2 and d(CTCTGACGTCAGAG)can be cross-linked by treatment with Fen-ton reagent. Pepsin digestion of the cross-linked product followed by separation andsequencing of the peptides showed that theoligonucleotide was covalently attached toSer-221 of RII. The location of this cross-linked residue readily explains the effect-ive quenching of Trp-226 fluorescence byDNA-binding. The presence of many hydropho-bic groups as well as the positively charg-ed sequence RRIIVKNNAKKRK in the vicinityof Ser-221 suggests possible modes of RIh(cA1P)2-DNA interaction at the bindingsite (USPHS GM 41610).

M-Pos17

SPATIAL DISTRIBUTION OF PROTEINS BOUND TODNA DETERMINED BY TIME-RESOLVED FLUORES-CENCE DEPOLARIZATION: APPLICATION TO DNAPOLYMERASE. D.P. Millar', C.R. Guestl,D.J.Allen2 and S-J. Benkovic2, lResearch Insti-tute of Scripps Clinic, La Jolla, CA 92037;2Penn State University, University Park, PA16802.

We demonstrate a new method for deter-mining the spatial distribution of a pro-tein bound to DNA in solution, based on thepicosecond time-resolved fluorescencedepolarization of an environmentally-sensi-tive dansyl probe covalently attached by alinker to a DNA base and positioned at aspecific site in the major groove. Cover-age of the probe by the protein increasesits excited-state lifetimes and stericallyrestricts the internal reorientation of theprobe. The bound protein is localized bythe dependence of the coverage on the probeposition on the DNA. Disorder of the com-plex produces a heterogeneous distributionof probe environments, resulting in a non-monotonic time-dependence of the depolari-zation. We have studied the complex of DNAand polymerase I (Klenow fragment), forwhich a crystal structure is unavailable.The complex was substantially disordered insolution. The effect of site-specificmutagenesis of the Klenow fragment on themode of DNA binding has been examined.M-Posl 19

STRUCTURAL DETAILS OF DNA IN THENUCLEOSOME. Thomas D. Tullius*, JeffreyHayes*, and Alan P. Wolffe#. *Department ofChemistry, The Johns Hopkins University,Baltimore, MD 21218. #Laboratory of MolecularBiology, NIDDK, NIH, Bethesda, MD 20892.We have used the technique of hydroxyl radicalfootprinting to investigate the structure of DNA inthe nucleosome formed with the 5S ribosomal RNAgene of Xenopus borealis. We find that the period-icity of cleavage by the hydroxyl radical is 10.0nucleotides per repeat, from either end to within twohelical turns of the dyad of the nucleosome. Aroundthe dyad the cleavage periodicity changes to approxi-mately 10.7 nucleotides per repeat. By cleavage ofthe same DNA molecule bound to a precipitate ofcalcium phosphate, we measured the helical period-icity of the 5S gene itself to be 10.4 base pairs perturn. These measurements were quantitatively eval-uated by fitting of a sine wave to the densitometerscans of the cleavage patterns, and by Fouriertransformation of the smoothed cleavage data. Wealso investigated the effect on DNA structure offorming a nucleosome on DNA designed to behighly curved. Implications of these measurementsfor the "linking number paradox", for the structuralbasis of phasing of nucleosomes on DNA, and forchanges in structure suffered by DNA uponincorporation into a nucleosome, will be discussed.



M-Posl 20

EFFECTS OF DNA, SOLVENTS AND A SINGLE-AMINOACID MUTATION ON THE PRESSURE-INDUCEDDISSOCIATION OF ARC REPRESSOR Jerson L.Silva, Cristina F. Silveira and Leila P.Silva. Dept9 Bioq. Universidade Federal doRio de Janeiro, RJ, Brasil. Arc repressoris a dimeric DNA binding protein that re

presses transcription from the Pant promo -

ter of bacteriophage P22 (Vershon, A.K.Younderian,P., Susskind, M.M., & Sauer, R.T(1985) J. Biol. Chem. 260, 12124. The equi-librium association reaction of arc repressor was studied by pressure-induced dissociation and by dilution. The dissociation was

measured by the decrease in the average e-

nergy of the the single tryptophan fluores-Icence. The dissociation constant (Kd) determined by pressure pertubation was the same

of that determined from the dilution curve(Kd=30nM). The subunit interaction was a-

bout 2.7 Kcal/mol more stable in the PL8 mu

tant. Glycerol (30%) conferred a stabiliza-tion of 2.0 Kcal/mol. Arc repressor boundto the plasmid pBR322 was more stable 1.3Kcal/mol than the unliganded protein. Howe-ver, DNA did not confer additional stabili-ty to PL8 dissociation. These data suggesta large coupling btween subunit-subunit andprotein-DNA interaction.Supported by FAPERJCNPq, FINEP.

M-Posl 22EFFECTS OF BASE MODIFICATIONS ONPROMOTER RECOGNITION BY T7 RNAPOLYMERASE. Martha D. Jaworski & Craig T.Martin, University of Massachusetts, Amherst, MA

Specific contacts between '7 RNA polymeraseand its promoter were probed by measuring theeffects of base modifications (dT-+ dU ordC- 5-methyl-dC) on transcription initiationkinetics. Positions of these substitutions werechosen to test and refine a model of polymerase-promoter binding in which RNA polymeraserecognizes one face of duplex DNA, including amajor groove and the two flanking minorgrooves (1). The above substitutions result in anoligonucleotide promoter in which a single methylgroup is either added to or removed from themajor groove of the duplex DNA. Steady statekinetic assays reveal values of Km ranging from50 nM (native template) to 700 nM, with thelargest effect occurring in the center of theproposed major groove recognition region(dT-+dU, -6C). There were no significant changesin kcat (10-20 liM/min), suggesting thatspecificity occurs at the level of binding and notcatalysis. These results further define polymerasecontacts with individual base functions in theDNA and show that methyl group interactionscontribute significantly to speciflc binding bypolymerase. Future studies with replacements ofdG-+dI and dA-+2,6-dia nopurine will determinethe role of m nor groove amino groups in

recognition by 17 RNA polymerase.1. D. K. Muller, C. T. Martin, & J. E. Coleman,

(1989) Biochemistry 28, 3306-3313.

M-Posl 21

The DNA Binding Properties of a Lac RepressorMutant That is Deficient in Dimer to TetramerAssociation. Amy Pickar, Elizabeth Jamison &Michael Brenowitz, Department of Biochemistry,The Albert Einstein College of Medicine, Bronx,NY 10461.The binding of wildtype Lac repressor (Lac+)

and a mutant repressor deficient in the dimer totetramer association (N. Mandal & S. Adhya,personal communication) to two sites on theDNA that are separated by 11 helical turns wasinvestigated using quantitative foot.print and mo-bility-shift assays. Binding of Lac' is highly coop-erative. DNase I hypersensitivity of the DNA be-tween the two binding sites is observed. This hy-persensitivi:ty is diagnostic of the formation of alooped complex", ie. a single Lac tetramerbridging the two sites. The "looped complex" is1.5 - 2.5 kcal/mol more stable than a tetramerbinding to a single site.

In contrast, the binding of the Lac repressormutant (Lacm) is non-cooperative and there is noevidence of "looped complex" formation. Bindingof Lacm was best-fit by a model where monomersu dimers and dimer + DNA = dimer-DNA withKdimer << Kdimer-DNA. The binding affinities ofLac' and Lac' to single binding-sites are compa-rable. These results support the role of the dimerto tetramer equilibrium in mediating the forma-tion of "looped complexes". (Supported by NIHGrant GM 39929).M-Posl 23DIFFERENTTAL PROMOTER BINDING AFFINITIES OFMONO- AND DI- LIGATED ESCHERICHIA COLI CRPAS MONITORED BY FLUORESCENCE ENERGYTRANSFER AND POLARIZATION. Tomasz Heydukand James C. Lee, Dept. of Biochemistry,St. Louis University School of Medicine,1402 S. Grand Blvd., St. Louis, MO 63104.

Structural and ligand bindingstudies have shown that CRP exhibits threeconformational states - free CRP and twocAMP-dependent states. In order to obtainvalid thermodynamic parameters to describethe linkages among cAMP and promoterbindings to CRP a simple fluorescenceprocedure to study DNA-protein interactionswas established. A 32 bp DNA fragment ofthe lac promoter containing the primarybinding site for CRP was labeled at the 5'end with a fluorescence probe. The bindingof CRP to this fragment can be convenientlyfollowed by monitoring the changes in an-isotropy or fluorescence energy transferfrom protein tryptophan residues to the DNAprobe. The results clearly demonstrated adifferential promoter binding affinitiesbetween the single- and double- ligatedCRP. The association constants for bindingof CRP-cAMP and CRP-(cAMP)2 to the promoterare 7.6 x 108 M-1 and < 1 x 106 M-1, respec-tively in 0.1 M KC1, pH 7.8, 23°C.

64a Biophysical Joumal voL 5Z 1990


M-Posl 24

19F-NMR OF T7 RNA POLYMERASE-DNAINrTERACTION.Fraydoon Rastinejad, Jean Ottand Ponzy Lu. Dept. Chemistry, Universityof Pennsylvania, Philadelphia, PA 19104.

By substituting 5-fluorodeoxyuracil (5-FdU) in place of thymine at specific sitesin promoter DNA sequences, 19F NMRsignals monitor the local changes uponbinding of T7 RNA polymerase andsubstrates. The 5-FdU substitutions do notimpair promoter function. The data fromsix substitutions has provided an NMRfootprint of the protein binding sitesthat can be compared with chemicalfootprints. The NMR data showsheterogeneity of chemical conformations inthe promoter as it interacts with theenzyme. At positions -6 and -10 thelargest perturbation of the fluorinechemical shift is observed, consistentwith the chemical data. Adjacent to thetranscription start-site, polymerasebinding induces a large inhomogeneousbroadening of the chemical shift,suggesting multiple small perturbations inthe DNA. The addition of GMP, as a deadend initiation substrate analogue, imposesa reduction of the structuralheterogeneity in this region, and causesobservable changes elsewhere in thepromoter. We are also using thetriphosphate analogues 5-F-UTP and 5-F-CTPto study defined RNA molecules and theirinteractions with proteins by 19F NMR.

M-Posl 26HUMAN TRIPLEX DNA-BINDING PROTEIN

PARTIAL PURIFICATION AND CHARACTERIZATION.Kiyama, R. and Camerini-Otero, D.NIH/NIDDK/GBB, Bethesda, MD 20892.

Polypurine-polypyrimidine sequences(poly(dA)-poly(dT), for example), which canform triplex DNA under physiological condi-tions, are often found in recombination hotspots and the promoter region of genes, buttheir biological functions remain unknown.

Here, we present evidence for aprotein(s) that binds to triplex DNA. Weused a gel shift assay for the detection ofthe protein. Substrates for the assay areoligonucleotides (dA)34 and (dT)34 whichcan make a triplex DNA (TAT triplex) in thepresence of MgL+ at neutral pH. After puri-fication of nuclear extracts from HeLacells by conventional column procedures, wehave obtained a fraction that contains aprotein(s) that binds specifically to theTAT triplex substrate but not to the ATduplex. To purify the protein further weused a triplex DNA-affinity column.

To confirm that the protein binds to thetriplex DNA, we recovered the band from agel and showed that the bound oligonucleo-tides are in the triplex form. With theaffinity-purified triplex-binding proteinfraction we can show that the protein hasa higher affinity to triplex DNA than toduplex DNA of the same sequence.

M-Po i25ON THE MECHANISM OF ASSEMBLY OF CHROMATININ VITRO. J. C. HANSEN & K. E. VAN HOLDE,DEPARTMENT OF BIOCHEMISTRY & BIOPHYSICS,OREGON STATE UNIVERSITY, CORVALLIS, ORWe have used analytical sedimentation to

study the in vitro assembly of nucleosomesonto a tandemly repeated sea urchin geneconstruct. At a histone/DNA ratio (r)=l.l,a 29S particle containing 12 nucleosomes isformed after dialysis from 2M NaCl into10 mM Tris/.25 mM EDTA buffer. However,at all r<l.l, we observe broad distribu-tions of <29S. Using r=l.l, we observe a19S particle after dialysis into lM NaCl.By 0.6 M NaCl, 85% of the templates sedi-ment at 26-29S, while the remaining 15%sediment at about 18S. In 0.3M NaCl, 15%of the material remains in the slowerboundary, while the fast boundary sedi-ments at 40S. These results indicate thatin vitro nucleosome assembly is a compli-cated noncooperative process apparentlyinvolving subnucleosomal particle formationby lM NaCl, followed by partial nucleosomeassembly and particle folding in moderatesalt, and climaxing with complete nucleo-some assembly and particle unfolding invery low salt.(SUPPORTED BY NIH GRANTS NOS. GM11719 ANDGM22916)

M-Posl 27

ADDITIVE AND NONADDITIVE EFFECTS OFBASE-PAIR SUBSTITUTIONS IN E. COLIPROMOTERS, Bruce A. Beutel and M. ThomasRecord, Jr., Departments of Chemistry andBiochemistry, University of Wisconsin-Madison

We have investigated the kinetics of interaction ofE. coli RNA polymerase (Ea70) holoenzyme withfour synthetic promoters, which differ in sequenceonly at two nearest-neighbor positions (-33,-32) inthe -35 region. Results of these in vitro studieshave been compared with in vivo measurements ofrelative promoter strength. We are interested in thequestion of whether effects of promoter sequencesubstitutions are context-independent (additive) orcontext-dependent (nonadditive). In vitro, base-pair substitutions at the -33 and -32 positions affectrate constants for both the bimolecular and unimol-ecular steps of the association mechanism, butthese substitutions have no effect on the dissocia-tion rate constant. Effects on the unimolecularstep(s) appear to be context-dependent, but the ef-fects on the overall bimolecular association rateconstant, as well as on the equilibrium constant foropen complex formation, appear to be context-independent. We propose a model to explain ourresults in terms of additive and nonadditive effectsof these sequence substitutions on the relative stan-dard free energies of the reactants, intermediates,and open complex.

Biophysical Joumal vol 5 7, 1990 65a

66a Biophysical Journal vol 57, 1990

M-Posl 28

Base Specificity of the SSB Proteln-ssPolynucleotide Binding Mode Transitlons.W.Bujalowski and T. M. Lohman, Department ofBiochemistry and Biophysics, Texas A&M University,College Station TX 77843

The E. coli SSB protein forms multiple complexeswith poly(dT), which differ by the number of nucleotidesoccluded per SSB tetramer; these are referred to as the(SSB)35, (SSB)56 and (SSB)65 binding modes (Lohman& Overman (1985) J. Biol.Chem.260, 3594; Bujalowski& Lohman (1986) Biochemistry25, 7799). We have nowexamined the salt-induced transitions among thesedifferent SSB tetramer - ssDNA binding modes for otherss polynucleotides, poly(rU), poly(rA), poly(dC) andpoly(dA) (pH,8.1, 250C). As we have previously shownfor SSB-poly(dT) complexes, poly(rU), poly(dA) andpoly(rA) form the (SSB)35 binding mode at low saltconcentration (< 5 mM NaCI). An increase in the saltconcentration induces the transition to the (SSB)56binding mode. However, the existence of anintermediate binding mode, (SSB)42, is observed; thismode was also observed in SSB-poly(dT) complexes at370C . In the case of poly(dC) the lowest site size bindingmode observed was (SSB)56. Studies of the binding ofdifferent oligonucleotides, dC(pC)69, dA(pA)69,dC(pC)34 and dA(pA)34 to the SSB tetramer reveals astrong dependence of the negative cooperativity amongssDNA binding sites upon the type of oligonucleotide.The correlation between the observed salt effect on theSSB-ss polynucleotide binding mode transitions and thebinding of oligonucleotides to the SSB tetramer will bediscussed.

M-Posl 30

THE SPACING OF NUCLEOSOMESON THE XENOPUS LAEVIS OOCYTE5S GENE. John D. Brantley1, and WilliamBlass2. 1Univ. of Kentucky. zUniv ofTennessee.The oocyte 5S gene of Xenopus laevis,

which exists as a set of tandemly repeatedunits, was solubilized by digestion withHind Ill. The resulting population of multi-mers was labeled by hybridization with anoligonucleotide probe and streptavidin col-loidal gold. Following dialysis into low saltspreading buffer, the samples were fixedwith glutaraldehyde, spread on hydrophiliccarbon coated grids, and shadowed. Label-ed molecules clearly showed a gold particleat one end. The separation of the nucleo-somes was measured from center to centerby tracing the curve of the chromatin on adigitizing tablet. The distribution of mea-surements was smoothed and deconvolutedusing the error distribution of the measure-ment process as the response function of thesystem modelled as a linear system. Theresulting distribution shows several periodicpeaks indicating that nucleosomes prefer tobe separated by multiples of 5.4 nm, orabout one half turn of the DNA helix.


M-Posl 29

ENHANCEMENT OF THE INCORPORATION OF DNAIN RECONSTITUTED SENDAI VIRAL ENVELOPES.Christopher Di Simone and John D.Baldeschwieler, California Institute ofTechnology, Pasadena, CA, 91125.

Triton X-100 reconstituted Sendaiviral envelopes have the ability toencapsulate nucleic acids. Thesevesicles have a radius of 100-200 nano-meters while a 5000 base plasmid may havean effective radius of 70-140 nm.Under normal reconstitution conditions,less than 1% of the total availableplasmid vector (5000+ bases) wasencapsulated. Addition of certain basicproteins ( polylysine, lysozyme, orprotamine) led to a greater than ten foldenhancement in encapsulation (6-10% DNAencapsulated). Experiments were designedto avoid confusion betwiyn associated andencapsulated material; P labeled DNAwas found to associate with the viralvesicles even after DNAse cleavage.

M-Pos131

DISSECTION OF THE 16S rRNA BINDINGSITE FOR RIBOSOMAL PROTEIN S4.

Amalia Sapag, Jailaxmi V. Vartikar,Anne-Marie Downes, David E. Draper.Department of Chemistry, Johns HopkinsUniversity, Baltimore, MD 21218.-

The ribosomal protein S4 from E. coliis one of the first to bind to 16S rRNA and isthus essential for the initiation of assemblyof 30S subunits. We have undertaken theidentification of specif'ic features requiredin the 16S rRNA for S4 recognition on a 463nt fragment (positions 39-500) which is theminimum domain capable of binding S4 withthe same affinity measured for the fulllength (1542 nt) rRNA molecule. We havesystematically deleted 11 hairpins found inthe secondary structure in order to evaluatetheir individual contribution to the bindingcapacity. Some may be removed withoutaltering intermolecular affinity, whereasexcision of others results in a several folddecrease in the binding constant. A set of N-and C-terminal deletions in the S4 proteinhas also been prepared and is being testedfor RNA recognition.


M-Posl 32

Structural Studies of the Cro Binding Site, Free and Boundto Cro Protein.Elisabeth Evertsz, Gerald Thomas, Warner Peticolas,University of Oregon, Chemistry Department

Raman spectra of the DNA binding site for crorepressor protein were obtained in the presence and absence ofbound cro protein. The 17 base pair fragment is identical tothq OR3 site except that the second base to the right of thecenter of pseudosymmetry is altered. Analysis of thespectrum of the free DNA indicates that the molecule existsin a B-like conformation with deviations from standard B-form occurring mainly in the bands assigned to A-Tvibrations. The spectrum of the bound DNA was obtained bysubtracting the spectrum of free cro from the spectrum of thecorpplex which was estimated to be 90% bound. The DNAundergoes significant structural changes upon binding to theprotein. most notable of these changes are a destacking of theG-C bases reflected by increases in the 1240, 1262, and 1320cm-1 bands. A decrease in the 1361 cm-1 band that occurshas also been assigned to a destacking in guanine bases.The appearance of the 705 cm-1 band and the decrease anddownshift of the 670 cm"1 band are consistent with theappearance of A-like character in the A-T region of thebinding site when the protein binds, however the spectraindicate that the entire binding site remains in a distorted B-like conformation. Other shifts in both intensity andposition cannot be assigned to characteristic changes inconformation and therefore must be attributed to the proteininfluencing the structure in a novel way.

M-Posl 34INVESTIGATION OF cAMP RECEPTOR PROTEIN

SECONDARY STRUCTURE BY RAMAN SPECTROSCOPY.Henry DeGrazia, James Harman', and Roger M.Wartell, School of Physics, Georgia Inst.Technology, Atlanta, GA, and tDept. of Chem-istry, Texas Tech Univ., Lubbock TX.

Raman spectroscopy twas employed to ex-

amine the secondary structure of the cAMPreceptor protein (CRP). Spectra were ob-tained over the range of 400- 1900 cm1

from solutions of CRP and from CRP:cAMPco-crystals. Estimates of the secondarystructure distribution were made by ana-

lyzing the amide I region of the spectra(1630- 1700 cm7l). CRP secondary structurewas essentially the same in pH 6 and 8.The amide I analyses indicated a structuraldistribution of 44% a-helix, 28% 8-strand,18% turn, and 10% undefined forCRP insolu-tion. Raman spectra of CRP:cAMP crystalsshowed several differences from spectra ofCRP in solution. Some differences were

attributed to CRP structural differences.Analysis of the amide I region indicated37% a-helix, 33% 8-strand, 17% turn and 12%undefined. Changes in the amide III regionand at 935 cm7'1 also indicated secondarystructure differences. A proposal that

cAMP induces a shift in CRP structure thatincludes the conversion of a-helix to8-strand is consistent with the Raman data.

M-Pos 133A SPECTROSCOPIC STUDY OF THEBINDING OF N-7- AND N-2-SUBSTITUTEDCAP ANALOGS TO HUMAN PROTEINSYNTHESIS INITIATION FACTOR 4ES.E. Carberr 1, E. Darzynkiewicz2,J. Ste!inski S.M. Tahara3, R.E.Rhoads & D.J. Gossl. lHunter Coll.of CUNY, NY; 2U. of Warsaw, Poland;3USC Sch. of Med., Los Angeles, CA;4U. of KY, Lexington, KY.

There is a correlation betweenthe affinity of N-7- and N-2-substituted 5'-terminal mRNA capanalogs to protein synthesis factoreIF-4E and their potency asinhibitors of protein synthesis.The pH and ionic strength dependentbinding of various N-7-substitutedderivatives have been used to mapthe eIF-4E binding site. Differ-ences in the binding of N-7-alkyl-and N-7-aryl-substituted capanalogs to eIF-4E appear to arisefrom favorable interactions of thephenyl ring with the guanosinemoiety, which may explain theenhanced recognition of the aryl-substituted cap analogs by eIF-4E.Grant Support: AHA, NSF, Min. Ed.Poland, Pol. Acad. Sci., NIH, ACS,M. Earl.y Trust.

M-Pos135CHROMATIN CONDENSATION IN THE FORESPORECOMPARTMENT OF SPORULATING CELLS OFBACILLUS SPECIES: VISUALIZATION BY FLUO-RESCENCE MICROSCOPY AND DIGITAL VIDEOIM-AGING-B. Setlow, P. Febbroriello, L.Nakhimovsky, D.E. Koppel and P. Setlow,Department of Biochemistry, UCONN HealthCenter, Farmington, CT 06032.

A key event in sporulation of Bacillusspecies is an unequal cell division, pro-ducing a smaller forespore and a largermother cell. Using the DNA stain DAPI wefind that at about the time of this divi-sion forespore chromatin becomes extremelycondensed. This condensation requiresproducts of s genes and the spoIIAC genebut not other spoII or spoIII loci.

Analysis of DNA distribution in thesporulating cell by digital videoimaginghas shown that the amount of DNA in thecondensation spot increases with time untilits value is approximately equal to the a-mount of DNA in the mother cell. Afterreaching this stage a partial decondensa-tion of chromatin occurs but the amount ofDNA in the forespore remains the same as inthe mother cell.

From these results we infer that thenumber of chromosomes in the mature sporeis the same as in the mother cell, and thatthere are significant changes in chromatinpacking during bacterial sporulation.



M-Posl 36

RESOLUTION OF HOLLIDAY JUNCTIONANALOGS BY T4 ENDONUCLEASE VII CANBE DIRECTED BY SUBSTRATE STRUCTURE.John E. Muellerl, Colin J. Newtonl, FrankJensch2, Borries Kemper2, Richard P.Cunninghaml, Neville R. Kallenbach3 and NadrianC. Seeman3. lDept. of Biology, SUNY/Albany,NY 12222, 2Institute of Genetics, University ofK6ln, FRG, 3Dept. of Chemistry, New YorkUniversity, NY, NY 10003.

Endonuclease VII is capable of resolvingHolliday recombination intermediates. We haveexplored its substrate requirements by usingimmobile analogs of Holliday junctions that lacksequence homology. We find that junctions whosedouble helical arms contain fewer than ninenucleotide pairs are not resolved. Scission ofsubstrates with arms symmetrically elongatedproduces cleavage of the lengthened arms. We useshamrock junction molecules formed from a singleoligonucleotide strand and either end-labeled orinternally labeled to confirm that the scissionproducts are those of coordinated resolutioncleavage, rather than nickdng of individual strands/.The relationship of the long arms to the cleavagedirection suggests that the portion of the enzymethat requires the minimum arm length interacts withthe pair of arms containing the 3' portion of thecrossover strands, on the bound surface of theantipaallel junction.

This research supported by GM-29554,CA-24101, GM-33346 (NIH) and SFB 74 (DFG).M-Posl 38VARIABILITY IN RHO-RNA COMPLEXES SEEN

BY CRYO-ELECTRON MICROSCOPYEd Gogol, Steve Seifried,

Hans Geiselmann and Pete von HippelInstitute of Molecular Biology

University of Oregon, Eugene, OR 97403

The E. coli transcription terminationfactor rho is a 47 kDa protein which bindsnascent mRNA tracts and brings about theirtermination and release in an ATP-dependent manner. Hydrodynamic measure-ments have established that, atappropriate protein and salt concentra-tions, rho monomers associate to formhexamers, in equilibrium with subpopula-tions of smaller oligomers. Hexamers ofrho have a latent ATPase activity which isactivated on binding RNA.

We have visualized rho by cryo-electronmicroscopy, both in the absence andpresence of oligomers of ribo-C. Imagesrecorded in the absence of RNA reveal twoclasses of structures, doughnut-shapedhexamers and, more frequently, arcs ofvariable length. Addition of oligomers ofribo-C long enough to span at least twomonomer binding sites results in a markedshift toward closed hexamers. Longeroligomers induce this structural transi-tion even more effectively by coopera-tively binding all six rho monomers.


M-Posl 37HIGH PRESSURE FLUORESCENCESTUDIES OF lac REPRESSOR REVEAL AROLE FOR THE SUBUNIT INTERFACE INREGULATING OPERATOR AFFINITYC. Royerl, A. Chakerian2 & K. Matthews2.1LUIUC, LFD, 110W. Green St.,LUana, IL 61801 &2Rlce Univ., Dept. Blochem., 6100 S. Main St..Houston, IX 77251.High pressure fluorescence studies were car-ried out on dansyl labeled lac repressor. Theaffinity between lac dimers was found to be4.5 nm, implying the involvement of thisequilibrium in measurements of apparentoperator affinity. The tetramer was found tobe destabilized at 21°C or at high pH. Inducerbinding had no effect on the tetramer at4.5°C, but resulted in a stabilization at 21°C.High pressure resulted in the dissociation ofthe repressor-operator complex at operatorconcentrations 105-fold over the dissociationconstant, indicative of a large volumechange for association. Finally, operatorbinding appears to decrease the affinitybetween dimers. These results bring tolight the coupling between subuiit interac-tions and ligand bindig. Such interactionsmay be important in determining the levelof repressor binding to multiple sites andpseudo-sites on looped DNA. (Supported byPHS-R29-GM39969 & PHS-RO1-GM22441.)

M-Posl 39STRUCTURAL STUDIES OF THE BAMHI RESTRICTIONENZYME. Teresa Strzelecka+, Lydia Dorner*,Ira Schildkraut* and Aneel Aggarwal+.+Dept. of Biochemistry and Mol. Biophysics,Columbia University, New York, NY 10032 and* New England Biolabs, Beverly, MA 01915.

Type II restriction enzymes are idealfor studying protein-DNA interactionsbecause of their high sequence specificityand striking variety. Large, well-orderedBamHI crystals, diffracting to 2.3 A, wereobtained from PEG solutions. These crys-tals occur in two forms: monoclinic (spacegroup C2, unit cell constants: a=76.24 A,b-46.0 A, c=69.4 I and P=110.5o) andorthorhombic (space group C2221, unit cellconstants: a=46.7 A, b-76.6 A and c-143.6A). In both crystal forms there is oneprotein monomer per asymmetric unit.Currently, we are searching for heavy atomderivatives of the enzyme.

We are also attempting cocrystallizationof the enzyme with a 12-bp DNA fragmentcontaining the BamHI recognition site(5'-GGATCC-3'). We have recently obtainedlarge, plate-like crystals, which we areexploring for the presence of DNA by X-rayand biochemical methods.


M-Posl 40

THE DECAY OFFLUORESCENCE ANISOTROPYOF ETHIDIUM BROMIDE BOUND TONUCLEOSOME CORE PARTICLES

David W. Brown, Elizabeth A.Winzeler, Louis J.Liberdni and Enoch W. Small

Department of Biochemistry & BiophysicsOregon State University, Corvallis, OR 97331-6503

The binding of ethidium bromide to the DNA ofnucleosome core particles is accompanied by a largeincrease in fluorescence lifetime from 1.6 ns to anaverage lifetime of about 22 ns, a change similar to thatobserved for the intercalative binding of etii um toDNA. The ethidium lifetime can be further enhancedup to an average of about 39 ns by substitutingdeuterium oxide for water. These relatively longlifetimes permit one to measure the decay of thefluorescence anisotropy out to relatively long times(greater than 350 ns for the deuterium oxide). Withsuch a decay one is able to clearly distinguish therelatively fast torsional motions of theDNA from therotational diffusion of the particle. The effects ofseveral solution-induced conformational changes onthe recovered rotational correlation times will bereported, as well as what these changes in correlationtimes imply about shape changes in the nucleosome.We will also use deconvolutions of the early time datato analyze the restricted motions of theDNA on theparticle. Supported by NIH grant GM-25663.

M-Posl 42FLUORESCENCE MICROSCOPY AND

CIRCULAR DICHROISM OF POLYLYSINE-DNA COMPLEXES

Eduardo Builes, Timothy W. Houseal, David A.Beach, and Carlos Bustamante.

Dept. Chemistry, University of New Mexico,Albuquerque, NM 87131.

We have used video fluorescence microscopy tostudy the condensabon of calf thymus and T2 DNAinduced by poly-D- and poly-L-lysine, andpolyethyleneglycol (PEG). The fluorescent dyeethidium bromide was used in these studies. Large,polynemic, superhelical filaments of T2 DNA wereobserved in samples prepared with 0.5 gg/mI poly-L-lysine, and some filaments displayed several levels ofsupercoiling. These superhelices were stable andmaintained their integrity after being mechanicallydisrupted. Toroids and spool-shapes were commonin samples prepared with 5.0 pg/ml poly-L- or poly-D-lysine. Clusters of condensates were observed insamples with PEG. The structures observed wereidentical to those observed by electron microscopy.Circular dichroism (CD) spectra were obtained forsolutions of calf thymus DNA with poly-L-lysine orPEG. A CD signal for DNA-PEG complexes could beobtained directly from the quantity examinedmicroscopically (4.0 p1). For DNA-poly-L-lysinecomplexes larger quantities were necessary, andaliquots of these were examined microscopically.This application of fluorescence microscopy providesa direct method of correlating structure of condensatewith CD spectrum. This technique will allow us tofollow, in real-time, the condensation process and tostudy the kinetics and dynamics of in-vitrocondensation of DNA.

M-Posl41

VISUALIZATION BY ELECTRON MICROSCOPY OFHOLLIDAY JUNCTIONS WITH BOUND REC A PROTEINSandra L. Shaner and Charles M. Radding,Dept. of Human Genetics, Yale Univ. Schoolof Medicine, New Haven, CT.

The reciprocal exchange of singlestrands during recombination between twohomologous DNA duplexes involves a branchedintermediate known as a Holliday junction[Holliday (1964) Genet. Res. 5, 282].Whereas many electron microscopic studieshave visualized, in the presence and ab-sence of bound protein, the reaction pro-ducts of recA protein-promoted asymmetricstrand exchange between a purely single-stranded (ss) DNA and a homologous duplex,the reaction products of symmetric strandexchange between two primarily duplex DNAmolecules have been less well studied.Electron microscopy has been performed pre-viously only on the deproteinized reactionproducts. Here, the Holliday structuresformed as a result of recA protein-promotedreciprocal strand exchange between a 1325bp duplex DNA with a 200 residue 5' ss tailand a partially homologous blunt-ended 2042bp duplex were examined by electron micros-copy in order to identify by direct visual-ization the location of bound recA protein.

M-Posl 43PROPERTIES OF FLUORESCENTLY LABELEDCATABOLITE ACTIVATOR PROTEIN AND RNAPOLYMERASE FROM E. COLI. MARK HENRYSINTON AND ARNOLD REVZIN, DEPARTMENTOF BIOCHEMISTRY, MICHIGAN STATEUNIVERSITY, EAST LANSING, MI 48824.

We have fluorescently labeled CAPand RNA polymerase to gain informationabout the structure of these proteins invarious types of transcription complexes.Each protein has, on average, a singlecovalently attached eosin group. Wehave (1) characterized the bindingaffinity of the labeled proteins forboth the wild type and UV5 lactose pro-moters, (2) compared the kinetic proper-ties of these complexes with those formedusing unlabeled proteins, and (3)assessed the ability of the labeledproteins to participate in the trans-cription process. The data imply thatthe presence of the eosin groupmodifies but does not eliminate theability of CAP or RNA polymerase tointeract with promoter regions in aphysiologically significant manner.



M-Poal 44Induced DNase I Hypersensitivity In OR3 bySimultaneous cl Repressor Binding to ORI and OR2Daniel Strahs and Michael Brenowitz. Department ofBiochemistry, The Albert Einstein College of Medicine,Bronx, New York 10461.

The protein cl repressor (cl) of phage X maintains thelysogenic state in infected E. coli cells by bindingcooperatively to three tandemly repeated binding sitesin the right operator of the phage X genome. When clrepressor binds simultaneously to sites OR1 and OR2transcription from the promoter PR is blocked andtranscription from the promoter PRM is enhanced. cl isbelieved to repress transcription initiation at PR bysterically blocking RNA polymerase (RNAP) binding andto enhance transcription initiation at PRM by protein-protein contacts between cl bound at OR2 and RNAP.

In vitro quantitative footprint titration studies of clbinding to OR-containing restriction fragments showthat DNase I hypersensitivity in site 0 3 is induced bythe simultaneous, and cooperative, binning of cl to OR1and OR2 The specificity of the hypersensitivity is shownby its absence upon binding cl to OR1-, OR2- and OR-2- mutant operators. Modelling of the hypersensitivityas a function of OR1 and OR2 occupancy suggests thatcooperative binding of cl can cause structural changesin the DNA. The implications of this result for themechanism of cooperative binding and transcriptionalenhancement at PRM will be discussed.(Supported by NIH Grants GM39929 and 5T32-GM-07128)


M-Posl 45INTERSUIUT CW NICATION IN c RECEPTOR PROTEIN FRN E.coti. Ewa Heyduk, Tomasz Heyduk, and James C. Lee. Dept.of Biochemistry, St. Louis University School of Medicine,1402 S. Grand Blvd., St. Louis, MO 63104.

Binding of cAMP to CRP exhibits a negative cooper-ative behavior. Therefore, there has to be communicationbetween the subunits interface which is formed almostentirely by a long alfa-heLix (C-helix). In search of aregion of this helix responsible for intersiuunit commun-ication two proteolytic fragments of CRP were generated andpurified. Ligand binding properties, conformational re-sponse to cAMP binding and global structural changes (usinganalytical gel chromatography) of these fragments wereprobed. The subtilisin fragment, which is devoided of 2/3of C-helix, binds cAMP in a non-cooperative way and under-goes a slight expansion when complexed with cAMP. Thechymotryptic fragment, which retains the whole C-helix,binds cAMP with high negative cooperativity and undergoes amore pronounced expansion. Intact protein, on the otherhand, exhibits slight contraction upon binding of cAMP.These results showed that the region of C-helix between Leu116 and Phe 136 is responsible for intersubunitcommunication, although it is not essential for dimerformation. They also suggest that ligand-induced conforma-tional change in CRP, which leads to the expression of thebiological function of the protein, can be a complexcoobination of changes in local (within a domain) structure,domain-domain and intersubunit interactions.

M-Pos1 46

THEORY FOR THE BINDING OF E.COLI SINGLESTRAND BINDING PROTEIN (SSB) TOSUPERCOILED DNA. J. B. Clendenning and J. M.Schurr, Department of Chemistry, University ofWashington, Seattle, WA 98195.

A recent theory for the binding of unwindingligands to supercoiled DNA is extended to includemultisite coverage. This is applied to a model forbinding SSB to supercoiled pBR322 in which the SSBbinds in pairs to the complementary strands of a meltedregion. Partition functions are evaluated using aniterative linearized matrix method. Values for thestability and cooperativity constants for duplex DNAand the intrinsic binding constant, binding-site size, andcooperativity parameter for the protein on single-strandDNA are all taken from the literature. Binding data arewell fitted by values within the reported ranges. Ourresults show that (1) SSB binding is driven largely bythe reduction of superhelical strain upon melting 34 bpper bound SSB pair, (2) SSB clusters into a singlecontiguous stack on each plasmid, (3) the stack lengthremains nearly constant over the observed concentrationrange, so the binding is virtually all-or-none, and (4) thelarge cooperativity due to opening the DNA iseffectively cancelled by the global anticooperativity thatresults from progressive unwinding, over the observedrange. These results are in complete accord withseveral different experimental observations.

LIPID-PROTEIN INTERACTIONS I

M-Posl 47AS MODEL OF THE MInICHCNDRIALVDAC CHANEL. C.A. Mannella, X.W. Guo, H.Chn, Wadsworth Center, NYS Dept. ofHealth and Depts. of Biomedical Sciencesand Physics, State Univ. of NY, Albany, NY.

The VDAC channel crystallizes in theplane of the outer membrane of Neurosporamitochorxdria by the action of phospholipaseA2. The geometry of the arrays can beexplained by a model in which the channelhas four binding sites on its periphery(A,B,C,D), which interact pairwise (A-B,C-D) with sites on adjacent channels. Thecrystal unit cell, which holds six channels,can acccmmodate one or two 30-kDa poly-peptides per channel. If the channel is amonomrer, most of the protein is needed toform the large transmembrane lumen, e.g.by folding into a beta-barrel as proposedby Guy (J. Bioenerg. Biomemb. 19:341). Thebackbone diameter of such a channel is 3.8nm based on projected density maps obtainedfrom cryoelectron microscopy of frozen-hydrated crystals. The sequence of the 20amino acids at the N-terminus is consistentwith an amphipathic helix. A model is pro-posed for a mnomrer channel in which gatingand ion selectivity involve movement ofthis helical "arm" between the bilayersurface and the channel lumen. (Supportedby NSF grant DMB-8613702.)

M-Pos149CALORIMETRIC STUDIES OF THEINTERACTION OF AN AMPHIPHILIC MODELPEPTIDE WITH PHOSPHATIDYLCHOLINEBILAYERS. Y. Zhang, R.N.A.H. Lewis, R.S.Hodges, and R.N. McElhaney, Department ofBiochemistry, University of Alberta, Edmonton,Alberta, Canada.

High sensitivity DSC has been applied tostudy the effect of an amphiphilic peptide (Lys2-Gly-Leu24-Lys2-Ala-amide) on the thermotropic behaviorof a series of diacyl-PC's with different hydrocarbonchain lengths. At high lipid:peptide ratios (>25-30lipids per peptide) the main ransition endotherms canbe resolved into two components, which we haveassigned to lipid rich (narrow component) and thepeptide rich domains (broad component). With andecrease in the lipid:peptide ratio there is a decreasein the transition temperatures of both componentsand a decrease in the total enthalpy change measured.We find that the peptide associated lipids make asignificant contribution to the total enthalpy change.Moreover, the acyl chain length dependence of thethermotropic properties of the peptide associatedlipids can be rationalized by a mismatch between thebilayer thickness and the length of the peptide.


M-Pos' 48

LIPID-PROTEIN INTERACTIONSMODULATES ADENOSINE DEAMINASEACTIVITY WHEN RECONSTITUTED INLIPOSOMES: EFFECTOFCHOLESTEROL

Stavanit C. Nemschitz, Valeria R. Caiolfa, Nurith Porat& Abraham H. Parola*, Department. of Chemistry,Ben-Gurion University of the Negev, Beer-Sheva,Israel 84 150

Adenosine deaminase (ADA) is a malignency markerwith a polymorphic structure: a small subunit, 45,000D,(SS-ADA) and an extrinsic membranal complexingprotein, 220,000D, (ADCP). ADCP was reconstitutedin liposomes (Caiolfa et al. (1989) Biophys J. 55:320a).Electron microscope micrographs verified our dynamiclight scattering studies of liposome size distribution.Retention of SS-ADA specific activity is evident whenbound to ADCP reconstituted in Asolectin or DMPCvesicles. Upon enrichment with cholesterol a 1.75 and 3fold increase in activity was observed with Asolectinand DMPC liposomes, respectivly. Excessiveincorporation of cholesterol, reduced SS-ADA activityin these vesicles. The quantitative differences observedbetween Asolectin and DMPC vesicles reflectsdifferences in lipid-protein interactions. Our results areintriguing when compared with ADCP-SS-ADAinteractions in solution which result rather in reducedADA activity. These results may explain the observedreduction in SS-ADA activity upon cell transformation.ADCP in buffer and in vesicles modulates SS-ADAactivity in an opposite fashion.

M-Posl 50EFFECTS OF PHOSPHOLIPID ACYL CHAIN AND PHVARIATION ON THE STRUCTURE OF BACTERIO-RHODOPSIN IN RECONSTITUTED VESICLES.Wen-Chang Huang and Barbara A. Lewis, Dept. ofChemistry, University of Wisconsin, Madison WI53706.

Bacteriorhodopsin (BR) is an Ideal modeltransmembrane protein. By performing solid stateNMR on reconstituted vesicles made withdelipidoted BR and a range of lipids, we areexploring the interactions of BR with its lipid andionic milieu. The goal is to elucidate the influensof hydrophobic and electrostatic forces on thestructure of this prototypical membrane protein.

When delipidated BR is reconstituted intophosphatidVlcholine (PC) vesicles, a blue-shiftedspecies (Xmax'm 490 nm) appears at alkaline pH.The apparent pKa for this transition is affectedboth by lipid acyl chain length and unsaturation.Solid-state 13x NMR studies of peptide-labeled BRin these vesicles are in progress. Using theapproach previously described (Lewis et.al.( 1985) 8iad,ristry 2A. 4671), lipid and pHeffects on the average peptide bond orientation(e.g., helix tilt angles) are determined.(Supported by NIH 0M38532.)

(Supported by the Medical Research Council ofCanada and the Alberta Heritage Foundation forMedical Research.)"

72. Biophysical Journal voL 57, 1990

MPosl 51

EFFECTS OF HEADGR9UP CHARGE ON THE ATPaseACTIVITIES AND Ca + BINDING PROPERTIES OFISOLATED CARDIAC SARCOLEAL VESICLESKenneth S. Leonards and Agnes Gardner,Dept. of Physiology, UCLA School of Medi-cine, CA 90024-1760

In recent studies we demonstrated thatthe free fatty acids produced by PLA2treatment can combi e with divalent ca-tions, such as Ca , to modify bothMg-ATPase and Na,K-ATPase activities inisolated cardiac sarcolemmal vesicles. Tofurther determine the relative importanceof headgroup charge in these interactionswe examined the effects of various concen-trations (0-100 pM) of a negatively chargedfatty acid analogue (SDS), its neutral an-alogue (lauryl acetate), and a positivelychlrged analogue (DDTMA) on the ATPase andCa binding properties of isolated cardiacsarcolemmal vesicles. Our results demon-stiate that headgroup charge, changes inCa + binding to the sarcolemmal surface,and inhibition of ATPase activity areclosely correlated in these vesicles, andsuggest that the surface properties of thesarcolemmal membrane may play a role in re-gulating Mg-ATPase and Na,K-ATPase func-tion. (Supported by an AHA(GLAA)Grant-in-Aid (843-Gl) the Laubisch Endow-ment, and a BRSG grant, UCLA)

M-Posl 53INFRARED STUDIES OF LIPID-PROTEIN INTER-ACTION IN PULMONARY SURFACTANT. Kim E.Reilly, Alan J. Mautone, and RichardMendelsohn, Department of Chemistry,Rutgers University, Newark,;NJ 07102.

SP-A, the major 29-36 kD class of sur-factant.protein, has been purified frombovine lung lavage and reconstituted intobinary lipid mixtures of DPPC-d62/DPPG.FT-IR melting curves of the lipid compo-nents revealed that high levels of SP-Ainduced an ordering of the phospholipidacyl chains. In contrast, a progressivedisordering of the acyl chains andreduction in the cooperativity of theirmelting event was noted upon reconstitu-tion of a mixture of the other surfactantproteins.

In addition to bulk transmissionexperiments, external reflection FT-IRwas used to examine monolayer surfactantfilms in situ at the air-water interface,as a function of surface pressure. Atsurface pressures which correspond tolarge lung volumes in vivo, the surfactantacyl chains exist mostly in the orderedconfiguration. The surface film under-goes a weakly cooperative transition asfunction of surface pressure which mimics(in terms of acyl chain order), a broadthermotropic phase transition in nativesurfactant.

UPID-PROTEIN INTERACTIONS I

M-Posl 52CALCIUM-INDEPENDENT BINDING OFPROTHROMBIN TO NEGATIVELY CHARGEDMEMBRANES. ISusan W. Tendian. 2Nancy L Thompson.& lBarry R. Lentz 1Biochemistry Dept. & 2ChemistryDept., Univ. of North Carolina, Chapel Hill, NC 27599.Intro, by Francine R Smith.A calcium-independent interaction of prothrombin withphosphatidylserine(PS)-containing membranes results inincreased fluid phase acyl chain order as detected by theanisotropy of diphenylhexatriene in small unilamellarvesicles (SUV). Binding has been quantitated using theprothrombin-induced shift in fluorescence anisotropy.The dissociation constant obtained by this method forprothrombin bound to 30/70 PS/phosphatidylcholine (PC)SUV was 2xl04 molar. The interaction was significantlyreduced with equivalently charged phosphatidylglycerol(PG)-containing membranes and was negligible with purePC membranes. The amino terminal third of prothrombin,fragment 1, displayed no such interaction with PS- or PG-containing membranes. The carboxy terminal 2/3 ofprothrombin, prethrombin 1, bound to PS/PC vesicles inthe same way as intact prothrombin, demonstrating thatprethrombin 1 is the region containing the calcium-in-dependent binding site. Total internal reflection fluor-escence microscopy measurements confirmed that PSsignificantly enhanced the binding of fluorescein-labelledprothrombin to planar PC membranes deposited on fusedsilica surfaces. Supported by USPHS(SCOR) Grant HL-26309 to BRL & NSF-PYI Grant DCB 8552986 to NLT.

M-Posl 54

THERMODYNAMICS OF CHOLERATOXIN BINDING TO VARIOUSDERIVATIVES OF ITS CELL SURFACERECEPTOR, GANGLIOSIDE GM1. MMasserini, A. Schbn, D. Xie and E. Freire,Department of Biology and The BiocalorimetryCenter, The Johns Hopkins University,Baltimore, MD 21218

The thermodynamics of the association ofcholera toxin to several derivatives of gangliosideGM1 (native GM1, Fucose-GM1 and gangliosideGM1 in which the fatty acid has been replaced byan acetyl group) incorporated into phospholipidvesicles has been measured by high sensitivityisothermal titration and differential scanningcalorimetry. The enthalpy of association of thecleaved oligosacharide region of ganglioside GM1to cholera toxin in solution is -165 kcal/mole oftoxin. On the other hand, the enthalpy ofassociation of the intact toxin to membrane boundganglioside is -100 kcal/mole and that of theisolated B subunit pentamer -110 kcallmole. Thedifference of approx. +60 kcal/mole most likelyarises from the direct interaction of the toxin withthe membrane surface since the enthalpy changesdo not exhibit a strong dependence on the headgroup or fatty acid modification of theganglioside. (Supported by NIH grants NS-24520 and RR-04328.)

LEPID-PROTEIN INTERACTIONS I

M-Posl 55

STOPPED FLOW KINETICS OF GALA-LIPIDINTERACTION. R. A. Parente. A. S. Verkman* && C. Szoka Jr., Schools of Pharmacy & *Medicine,University of California, San Francisco, CA.

The rate of pH driven association of the syntheticamphipathic peptide, GALA, with unilamellar phos-phatidylcholine (PC) vesicles has been investigatedusing stopped-flow fluorescence. Association wasfollowed by fluorescence energy transfer from the N-terminal Trp of GALA to an NBD-lipid probeincorporated in the PC vesicles. An increase influorescence was measured when GALA associatedwith vesicles at pH 5. At 1 mM POPC the relaxationtime for peptide-vesicle association was independentof [GALA] up to 40 uM and equaled 23±2 ms. Theinitial stages of lipid-peptide association can bedescribed by the rate equation, L + nP - L- Pn, whereunder conditions of [L] >>[P], the process is a pseudofirst order reaction. When the pH of the premixedvesicle-peptide complex was raised from 5 to 7.5, thedissociation rate was an order of magnitude slowerthan the association rate.

The initial rate of GALA-induced leakage fromvesicles was also studied on the millisecond timescaleby stopped-flow fluorescence. A lag time in the initialrate of leakage was observed. This lag in the onset ofleakage increases from 0.2 to 5 s as the lipid to peptideratio is increased from 50:1 to 15000:1. These resultsare consistent with a model in which GALA rapidlyassociates with the vesicle and aggregates in themembrane to form a pore or channel.

M-Posl 57INTERACTION OF SYNTHETIC MAGAININSAND POLYMYXIN B WITH LPS AND OUTERMEMBRANES OF POLYMYXIN-RESISTANTAND -SENSITIVE STRAINS OF SALMONELLATYPHIMURIUM. Fazale Rana*, Elizabeth Maciast,Malcolm Modrzakowskit and Jack Blazyk* (Intr. byP. Sullivan), Departments of *Chemistry andtZoological and Biomedical Sciences, and College ofOsteopathic Medicine, Ohio University, Athens, OH45701.

Lipopolysaccharide (LPS) in the outer membrane ofSalmonella typhimurium is phosphorylated at num-erous sites with either monophosphate or diphosphatemonoesters or diesters. The ability of syntheticmagainins and polymyxin B to bind to and disorderLPS in the outer membranes of polymyxin-resistant(SH5357) and -sensitive (SH5014) strains (gifts fromE. McGroarty) is related to the nature of thesephosphorylation sites. The effects of magainin 2, themore potent analog, [Ala13"18]-magainin 2, andpolymyxin B on the thermotropic behavior of LPSand isolated outer membranes from the two strainswas monitored by FT-IR spectroscopy. The moleratios of phosphatidylethanolamine to LPS in theouter membranes of each strain and the nature of thephosphate groups on LPS were determined by high-resolution 31P NMR spectroscopy. The degree oflipid disordering and lipid composition are correlatedwith the bactericidal potency of the three peptidestoward the two Salmonella strains.


M-Posl 56

CHOLESTEROL ENRICHMENT INCREASES CAV +AND K+ FLUXES AND DECREASES MEMBRANEFLUIDITY IN ARTERIAL SMOOTH MUSCLE. T.N.Tulenko, M.M. Gleason and M.S. Medow. MedicalCollege of Penna. Phila, PA. (Intro. by R.S.Moreland)

The cholesterol :phospholipid molar ratio(C/PL) in the plasma membrane regulates thephysical state (fluidity) of the phospholipidbilayer. The relationship between membrane Ccontent, and K+ and Ca++ movements andfluorescence anisotropy were investigated incultured rabbit aortic smooth muscle cells (SMC).SMC were incubated in media ± 3 types of C/PLliposomes: C-rich (2:1 C/PL), C-poor (0.5:1C/PL) and C-free (0:1 C/PL) with constant PL(550 gg/ml media). Incubation with C-richliposomes; 1) increased cellular C mass with nochange in PL content resulting in an increase inC/PL (p<.001) and 2) increased SMC microsomal Cmass with no change in PL content resulting in aincrease in C/PL (p<.05). C enrichment increasedbasal Ca++ influx and Ca++ and K+ effluxes. Netcellular C correlated with increased basal Ca"+influx (r=.762) and Ca++ and K+ efflux rates(r=.923). C enrichment of SMC increasedfluorescence anisotropy. These results suggest thatthe SMC plasma membrane cholesterol content issensitive to enrichment and depletion, and thatchanges in either direction alter transmembraneCa++ and K+ movements and membrane fluidity.

M-Posl 58ROLE OF LPS IN THE INTERACTIONS OFMAGAININ WITH THE S. TYPHIMURIUM CELLENVELOPE. Fazale Rana*$, Elizabeth Maciastt'Catherine Sultany+, Malcolm Modrzakowskit* andJack Blazyk*$, Departments of *Chemistry andtZoological and Biomedical Sciences, $College ofOsteopathic Medicine and +University Research andInstrumentation Center, Ohio University, Athens, OH45701.

Smooth wild-type Salmonella typhimurium and threerough strains possessing Ra, Rd, and Re chemotypeLPS show susceptibility to the antimicrobial peptide,magainin 2, in the following order: Re > Rd, 2 Ra >smooth. The fluidizing effect of magainin on theouter membranes and lipopolysaccharides (LPS) ofthe four organisms was examined by FT-IRspectroscopy. The extent of disordering induced bymagainin in the outer membranes is greatest in thesmooth and least in the Re strain. Heterogeneity ofthe LPS sugar phosphorylation sites, net charge onthe LPS molecules and the relative amounts of LPSand phosphatidylethanolamine (PE) in the outermembranes of each strain were determined by high-resolution 31P NMR spectroscopy. The concentrationof LPS in the outer monolayer and the anionicity ofLPS from the different strains appear to beresponsible for differences in magainin binding. Wepostulate that LPS in the outer membrane may act as amolecular sponge for the cationic peptide, inhibitingits lethal attack on the plasma membrane.

74a Biophysical Joumal vol 57, 1990

M-Poul 59THE EFFECT OF SIGNAL PEPTIDES ONMEMBRANE LIPID ORGANIZATION

J.A. Killian, J. Bijvelt, AJ. Verkleij and B. de Kruijff,CBLE, Padualaan 8, 3584 CH Utrecht, The Netherlands

To get insight into the possible role of signal sequences inprotein-translocation we investigated the effect of severalsynthetic signal peptides on lipid structure in modelmembranes of phosphatidylethanolamine and phosphatidyl-glycerol mimicking the lipid composition of the E. coliinner membrane. Using lp NMR and freeze fractureelectron microscopy it is demonstrated that addition of thesignal peptide of the E. coli outer membrane protein PhoEto these model membranes even at very low concentrations(±1/3000 molar ratio of peptide to lipid) strongly promotesthe formation of type II non-bilayer lipid structures.Furthermore, using a negatively charged mutant signalpeptide, a correlation was found between the effect on lipidstructure and in vivo functioning of the correspondingprecursor protein. The changes in lipid structure uponaddition of the PhoE wild type signal peptide and othersignal peptides are found to be similar to those induced inthe pure lipid system upon incubation at elevatedtemperatres.It is proposed that signal sequences may act by inducingthe local and transient formation of type II non-bilayer lipidstructures that allow precursor proteins to cross themembrane.

M-Posl 61

FORMATION OF METARHODOPSIN II ANDACTIATION OF 0v BY RHODOPSIN-CONTAININGDMPC VESICLES.Drake C Mitchell Julia Klbelbek and Burton J. LitmanDepatment of Biochei,s Univrsi f Vginia, SchoolfMedicn Charloteville, Vuuinia, 22908.

Formation of meta II and rho were measuredindependently in DMPC (T, = 23C) and POPC (T, = -2C) vesicles, using laser flash photolysis and G, activationmeasurements, respectively. At 30 and 37C the meta I-meta II equilibrium constants are 0.4 and 0.75 in DMPC,2.0 and 2.9 in POPC, respectively. The presence of G,enhances production of meta II by binding to meta II inthe absence of GTP. In POPC and DMPC, at 30 and 37C,the degree of enhancement corresponds to Gv binding toall available photolyzed rhodopsin molecules, but theapparent rate of binding in POPC is approximately 20times greater than that in DMPC. In DMPC vesicles at37C all the Gv-bound GDP exchanged for GMPPNP, anonhydrolyzable GTP analogue, during a 5 minuteexposure to room light, while at SC less than 5% of theG, had undergone exchange. In contrast, in POPCmaximal exchange was obtained at both 5 and 37C Theseresults show that photoactivation of rhodopsin,incorporated into a DMPC bilayer, produces meta II andfunctionally competent rho . However, the dependenceof the functional coupling of rhodopsin and Gv on thebulk microenvironment provided by the phospholipidbilayer is demonstrated by the minimal activation of G,below the T, of DMPC. In sharp contrast, maximalactivation was observed in the POPC vesicles at 5 and37C; temperatures which are above the T. of POPC(supported by NIH grant EY00548).


M-Posl 60A QALITATIVE AND QUANTITATIVE STUDY ON THE

BINDING OF CYTOCHROME C TO A DIOLEOYLPHOSPHATIDYLCHOLINEMONOLAYER SPREAD AT THE AIR-WATER INTERFACE

Fran;ois LAMUAR9E1, Jean-Joseph MAXPierre LAVIGNE et Roger M. LEBLANC3

1Centre de recherche et de d6veloppement sur les alimentsAgriculture Canada3600, boulevard Casavant OuestSaint-Hyacinthe (Qu6bec) Canada

2D6partement de chimie-biologieand

3Centre de recherche en photobiophysiqueUniversit6 du Qu6bec I Trois-RivieresCase postale 500Trois-Rivi&res (QuLbec) Canada

The monolayer technique has been widely used to study thelipid-protein interactions in biomembrane models. Thebinding of cytochrome c (cyt c) to different neutral andcharged lipids has been previously reported by differentauthors. The general apWroach to characterize thesesystems is the measurement of adsorption kinetics. Theseexperiments are generally time consuming and they givelittLe quantitative information. Ue have undertaken thestudy of cyt c incorporation into a dioleoylphosphatidyl-choline (DOPC) monolayer using a different approach. Thelipid-protein system was characterized from surfacepressure-moLecuLar area isotherms obtained for a dynamic-ally compressed-expanded fiLm. From these measurements,surface pressure limits for adsorption/desoption areunambiguously determined. Furthermore, the free energy ofincorporation, desorption and stabilization can bedetermined from those isotherms. A characterization oftransferred DOPC-cyt c films by electron microscopycomplete this study.

M-Posl 62CHOL STEROL INCREASES THE STABILITY OFRHODOPSIN IN EGG PC VESICLES TOTHERMALLY INDUCED UNFOLDING.Andrew P Mone and Burton J. Litman, Department ofBiochemisty, Univesiy of Vuviia, School ofMedicine, Charlotesville, Vquina 22908.

Studies have been carried out to determine the effectof the addition of cholesterol to egg phosphatidylcholine(PC) vesicles on the thermal stability of an incorporatedintegral membrane protein. Tb this end calorimetric,spectrophotometric and spectropolarimetric measurementsof rhodopsin's thermal unfolding in egg PC vesiclescontaining 0% and 30% cholesterol were made. Data fromall three types of measurements concur and reveal amidtransition temperature 3 degrees higher for thecholesterol containing membranes. The maximum of theexcess heat capacity function occurs at 72C and 68.5Crespectively, for those membranes with and withoutcholesterol. The calorimetric enthalpy of unfolding is130± 10 kcaUlmole for rhodopsin in both preparationsconsistant with similiar folded states and similiar unfoldedstates in the 0% and 30% cholesterol membranes. As theprotein unfolding is irreversible, a kinetic study of thechange in an observable is appropriate. Fitting the lossof rhodopsin's 500nm absorbance to a single exponentialfunction yields rate constants of 0.020 min-1 for 30%cholesterol membranes and 0.040 min-I for 0% cholesterolmembranes at 65C. These results suggest an increasedactivation energy for the unfolding of rhodopsin in thecholesterol containing membranes due to the orderingeffect of cholesterol on the PC acyl chains. (supported byNIH grant EY00548).


M-Posl 63HDL, APO Al AND AMPHIPATHIC PEPTIDESSTABILIZE LIPOSOMES COMPOSED OF DIOLEOYL-PHOSPHATIDYLETHANOLAMINE (DOPE) AND OLEICACID (OA). Dexi Liu and Leaf Huang, Dept.of Biochem., Univ. of Tennessee, Knoxville,TN 37996 and Jere P. Segrest and G.M. Anan-tharamaiah, Dept. of Med., Univ. of AlabamaSchool of Med., Birmingham, AL 35294.

Small unilamellar liposomes composed ofDOPE and OA are stabilized by incubationwith normal human serum or plasma (Liu andHuang, Biochem (1989) 28, 7700-7707).Systematic studies on interaction ofpurified serum proteins with this typeliposomes showed that albumin destabilizesliposomes by extracting OA from liposomes.However, HDL and, to some extent, VLDLshowed a rapid stabilization activityagainst the lytic effect of albumin.Purified Apo Al, showed comparable stabili-zation activity as HDL. Furthermore,synthetic peptides resembling the amphipa-thic helices found in Apo Al also showedstrong stabilization activity. These dataindicate that Apo Al plays a major role inhuman serum or plasma for the liposomestabilization activity. The stabilizationactivity is probably through the interac-tions of the amphipathic helices of Apo Alwith the hydrophobic voids on the outersurfaces of liposomes. Supported by NIHgrants AI25834, CA24553, and 5-POl-HL34343.

M-Posl 65

EFFECT OF PHOSPHATIDYLCHOUNE (PC) ON THEORIENTATION OF 3-HYDROXYBUTYRATE DEHYDRO-GENASE (BDH) IN PHOSPHOUPID (PL) VESICLES.Lauraine A. Dalton, J. Oliver Mcintyre and Sidney Fleischer.Vanderbilt University, Nashville, Th 37235.BDH Is a membrane-bound enzyme with an absolute

requirement for PL for enzymic activity. PC activatesoptimally and phosphatidylethanolamine (PE) activatespartially (see Mcintyre et a/., this volume). BDH has beenderivatized at a single active-center sulhydryl (SHI1) withthe spin label 2,2,6,6-tetramethylmaleimidopiperidinyl-1 -

oxyl (MSL). The nitroodde (NO) of MSL is 8 A from SH1.MSL-BDH can be inserted unidirectionally into PL vesicles(BDH-V) containing di (DPG) and eihrPC or PE. Uneshapes of EPR and ST-EPR spectra ofMSL-BDH were similar in the presence vs absence of PC,indicating comparable rotational mobility. Accessibility ofthe NO of MSL-BDH to paramagnetic Gd3+ was comparedfor BDH-V containing DPG and either PC or PE. Dipolarrelaxation of NO by Gd3+ bound to DPG at the surfaceof the bilayer permits calculation of radial distance. ForMSL-BDH-V in DPG/PE, the distance of closest approachof Gd3+ to the NO (m9 A) is much less than for MSL-BDH-V in DPG/PC [17 A, Dalton et al., Biochemistry 26, 2117(1987)]. The shorter distance between bound Gd3+ andNO in MSL-BDH-PE vs MSL-BDH-PC indicates that PCinduces a conformational change in BDH resulting in achange in orientation of either the MSL with respect toBDH and/or BDH with respect to the PL bilayer. Thenature of the PL determines the distance of the NO fromthe membrane surface but not the motion of BDH.[Tennessee Heart Assn. (JOM) and NIH DK 14632 (SF)].


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CHARACTERIZATION OF PHOSPHOLLPID-AMPHIPATHIC PEPTIDE COMPLEXESW. W. Mantulinl & J. T. Sparrow2 1UIUC,LFD, 1110 W. Green St., Urbana, IL 61801 & 2BaylorCollege of Medicine, Houston, TX 77030.The synthetic, 20 residue, lipid-associatingpeptide LAP-20 (VSSLLSSLKEYWSSLKESFS)associates with dimyristoyl phosphatidyl-choline (DMPC) to form complexes ofdefined stoichiometry and size. In solution,LAP-20 is a random coil, however, in thepresence of DMPC, it acquires amphipathichelical properties and is capable of activatinglecithin:cholesterol acyltransferase (Ponsinet al., 1986, J.Biol.Chem. 261:9202). We have pre-pared DMPC/LAP-20 complexes at a moleratio of 100:1 with a 50 nm pore membraneextruder (Lipex Biomembranes, Inc.) Nega-tive stain electron microscopy and dynamiclight scattering measurements show thatthese particles are asymmetric in size andshape. In the complexes, the tryptophan flu-orescence spectrum is blue shifted to 324 nmand the lifetime is shorter. Analysis of life-time data by lorentzian distribution shows adecrease in the lifetime with increasingtemperature. The distribution width corre-lates with the physical state of the DMPC.(PHS-P41-RR03155.)

M-Posl 66

PHO0SPHOIJ:PID INCORPORATION OF P32 INOPRIMAIRY MYCOIUBE CUIJTURES OF DYSTIUPHICMX AND WNIUL NJSCIR. Judy E. Andrson,

artent of Anatany, University ofManitoba, Canada. R3E 0W3.

Ambrane phospholipids (PUP) anchiorcytoskeletal proteins, and have been usedas a marker for fusion processes duringir ogenesis. The DMD gene product, dystro-phin is thought to be critical in preven-ting sarcolemmal instability during muscleactivation. However, dystrophin deficiencyfran nxx muscle does not prevent regenera-tion fram early dystrophic degeneration.Incorporation of P-32 fran orthcphosphoricacid into PIP was studied fran chloroform:methanol extracted primary cultures ofmuscle fram newborn xrdx and ccntrol mice,after the appearance of rryofibrils in themyotubes. P-32 incorporation into mdx PUPwas significantly increased into phospha-tidyl ethanolamine and was decreased inlysophosphatidyl choline and phosphatidylserine/phosphatidyl inositol fractionsisolated fran TILC plates as ccnpared withthe sane fractions fran control PLP.

Supported by the Manitoba Health ResearchCoucil


MPosl 67

CONFORMATIONAL DYNAMICS OF MELITT-l-INAND [Ala-14]-MELITTIN BY NMR AND AMIDEEXCHANGE, AND SIGNIFICANCE FORMELITITIN'S MEMBRANE ACTIVITIES.Christopher Dempsey, Renzo Bazzo and Iain D.Campbell (Intro by Steven Smith); Biochemistry Dept.,Oxford University.

The contribution of the central proline residue tothe conformational and dynamic properties, andmembrane activities of melittin, has been determinedusing a synthetic analogue, [Ala- 14]-melittin, in whichthe proline residue has been replaced by alanine. ThepH-dependence of single amide exchange from [Ala-14]-melittin in methanol has been determined by 'HNMR and compared with similar data for melittin[Biochem. 27 6893 (1988)]. The replacement of prolineby alanine results in a marked stabilization of themelittin helix by ten-fold in the N and C terminalregions and by up to 100-fold in the central turn ofhelix containing Pro- 14 of melittin. Local equilibriumconstants for individual hydrogen-bond-breakingfluctuations are calculated and the data analysed interms of models for helix fluctuations. Thereplacement of proline has marked effects on thehaemolytic and voltage-dependent channel activities ofmelittin in membranes and these effects areinterpreted in terms of the altered conformational anddynamic properties of the analogue.

M-Posl 69THE ROLE OF POLARIZATION ENERGYIN FREE ENERGY CALCULATIONS.

K. Ramnarayan, B.G. Rao and U.C. Singh(Intro. by Dr. V.N. Balaji)

Department of Molecular BiologyResearch Institute of Scripps Clinic10666 North Torrey Pines Road

La Jolla, CA 92037

A detailed implementation of the polariza-tion energy and its derivatives into a

molecular dynamics program (AMBER, version3.3) is described. In order to examine theeffect of the polarization energy on thecalculated free energy differences usingfree energy perturbation method, we havecomputed the differences in free energy ofsolvation of normal alkanes, tetraalkyl-methane, tetraalkylammonium ions and some

closed shell ions in water. The calculatedfree energy differences are in reasonableagreement with the experimental values.The pattern of the free energy changecomputed is compared with the results ofour earlier simulations where the polariza-tion energy was not included in the calculation. It is found that, in the majorityof the cases, the polarization energy con-

tribution to the free energy change isadditive.

STRUCTURAL & MOLECULAR DYNAMICS

M-Posl 68MODELLING OF ELECTROPORATION INMEMBRANES BY THE NUCLEATION THEORY INCONDENSED SYSTEMS. M. Toner and E.G. Cravalho,Harvard University-Massachusetts Institute ofTechnology Division of Health Sciences and Technology,Cambridge, MA 02139. (Intro.by DrR.C. Lee).The rate of production of critical size pores resulting inmembrane rupture in the presence of an appliedtransmembrane potential is modelled based on the theoryof nucleation in condensed systems. The steady-state rateof critical pore formation was described by I - O wN*;where 0 is the number of water molecules in contactwith the critical pore, w is the molecular jump rate, andN* is the equilibrium number of pores of critical size perunit membrane area (Am). The time constant to cause themembrane rupture was estimated to be ' = 1/JAm. Forartificial bilayers, the estimate of 'z was in reasonableagreement with experiments. Irreversible mechanicalbreakdown time drops from - 25,000 ms at 200 mV to0.024 ms at 240 mV. It was also shown that the dynamicsof pore formation could be modelled only by includingthe transient behavior. At 240 mV, the time to reach 63%of the steady-state pore formation rate was about threeorders-of-magnitude less than the estimated membranerupture time. However, at a slightly higher membranepotential of 270 mV, the transient time is one order-of-magnitude longer than the rupture time and could not beignored. The analysis from this work was intended toestablish the basis for a more elaborate modelling oftransient electroporation phenomenon in membranesusing the principles of the transient nucleation theory.

M-Posl 70ROTATIONAL DYNAMICS OF FcE RECEPTORS ONINDIVIDUAL 2H3-RBL CELLS STUDIED BYPOLARIZED FLUORESCENCE DEPLETION. B.G.Barisas, D.A. Roess, I. Pecht, and N.A.Rahman, Colorado State University, Ft.Collins, CO 80523.

Polarized fluorescence depletion (PFD)has been used to measure the rotation offunctional membrane proteins on individ-ual, microscopically selected cells underphysiological conditions. Combining longtriplet-state probe lifetimes with thesensitivity of fluorescence detectionrenders measurable rotational correlationtimes X from < 10 As to > lms. X for FcEreceptors (FcER) on the surface of 2H3-RBLcells is 79.9 ± 4.4 Asec and 83.6 ± 6.7lssec at 4°C when labeled with eosin conju-gates of IgE and of F4 anti-FcER Fab frag-ments, respectively. These values agreewith the known 100 kDa receptor size andwith previous phosphorescence studies.When labeled with intact F4 antibody, Xfor FcER increases about 2-fold to 170.8 +6.5 gsec, consistent with FcER dimer for-mation on the plasma membrane as deter-mined independently. These studies demon-strate the utility of PFD measurements inassessing size and association of membraneproteins on individual cells. Supportedby NIH grants AI-21873 and AI-26621 (BGB).


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PHOSPHORESCENCE QUENCHING OFPROTEINS ATROOM TEMPERATURE BYADDED SMALL MOLECULES. S. Papp, W. W.Wrighl, S. W. Englander, J. M. Vanderkooi & C S.Owen . Dept. of Biochemistry & Biophysics, School ofMedicine, Univ. of Pennsylvania, Philadelphia PA19104 & Biochemistry Dept., School of Medicine,Jefferson Univ., Philadelphia PA 19103Tryptophan phosphorescence lifetimes for proteins insolution at room temperature vary from about 20 usecto 2 sec. In a study of ten proteins (thermolysin,parvalbumin, nbonuclease Ti, nuclease, alcohol andglyceraldehyde-3-phosphate dehydrogenase, aldolase,pronase, azurin, alkaline phosphatase), long lifetimecorrelates with rigid environments in alpha helix orbeta sheets. Added small molecule quenchers canreduce lifetimes. Against a given protein, thequenching constants found for different quenchers(nitrite, ethanethiol, nicotinamide, azide) areremarkably constant. Through the sequence of groteinslistqd, thf quenching constant varies from 10 to 101M- sece , decreasing exponentially with the distance ofthe indole from the protein surface. Phosphorescencequenching may therefore be used to measure thedistance of tryptophans in proteins from the aqueoussurface without covalent modification of the protein. Itis also interesting that the quenching mechanismappears to involve long range electron transfer.(Supported by NIH GM 34448 & DK 11295)

M-Pos1 73FLUORESCENCE EMISSION AND ANISOTROPY DECAYOF THE (+)-anti-BENZOtAIPYRENE DIOLEPOXIDE ADDUCT IN POLY (dg-dC)-(dG-dC)A. Graslunda S. Eriksson , B. Norden andB. Jernstrom .

aDept. of Medical Biochemistry and Bio-physics,bUniversity of UmeA, S-901 87 UmeA,Sweden, Dept. of Physical Chemistry,Chalmers University of Technology,S-412 96 Gothenburg, Sweden and Dept. ofToxicology, Karolinska Institutet,S-104 01 Stockholm, Sweden.(+)-anti-7,8-dihydrodiol-9,10-epoxy-benzo-Eaj pyrene (BPDE), a strongly carcinogenicmetabolite of benzo[a)pyrene binds specif-ically to guanine in double stranded DNA.The fluorescence of the BPDE adduct inpoly (dG-dC) is characterized by severallifetimes. Part of the population givesrise to excimer fluorescence with a diff-erent excitation profile compared to themonomer. Using synchrotron radiation asexcitation we have studied the fluorescenceemission and anisotropy decay propertiesof both monomer and excimer populations.The anisotropy decay curves may be des-cribed by only one decay time. This wasfound to be around 5 ns for monomer and1 ns for excimer emission, indicating amore rapid mobility of the BPDE's bound asexcimer forming adducts.


M-Posl 72

HYDROPHOBIC AQUEOUS NANOCOLLOIDSAS MODEL FOR AGGREGATION OF PROTEINSA. Blum (Intro. by D. A. Goldstein)

Water insoluble heterocyclic aromaticcompounds can be distributed in aqueousmedium to form a stable suspension ofsubmicron, spherical particles. Suchtranslucent suspensions have narrowmonodisperse size distribution and arestable for more than one year. Theeffects of ionic strength, pH,temperature and hydrophobic interactionson the filtration, settling, aggregationand aging behavior of the nanocolloidalsols represents a close analogy to thebehavior of protein solutions. The time-dependent transition from reversible toirreversible aggregates observed fornanocolloidal sols can mimic the non-specific interactions in proteinaggregation. Due to chemical homogeneityof nanocolloidal particles surface thereis good chance for better simulation ofnon-linear aggregation effects incomputer dynamics. The addition ofproper components allows for modellingof protein-lipid, protein-DNA andprotein-sugar interactions. Use of nano-colloidal sols as experimental "testingboards" for computer simulations ofprotein interactions is proposed.

M-Posl 74MOLECULAR MECHANICS/DYNAMICS OF METAL IONBINDING: PARAMETER DEVELOPMENT FOR ALKA-LINE EARTH AND LANTHANIDE IONS.William DeW. Horrocks, Jr. Department ofChemistry, The Pennsylvania State Univ.,University Park, PA 16802The recent success of the molecular me-chanics/dynamics approach to the under-standing of the structure, energetics anddynamics of proteins has prompted us toapply these techniques to metallobiomole-cules. Non-bonded parameters have beendeveloped for ions whose bonding is largelyelectrostatic. Our approach is to use theCHARMM program to stimulate (with full 3-dimensional periodicity) x-ray structuresof complexes involving biologically rele-vant ligands and to perform molecular dy-namics calculations on ions in solutionwith explicit TIP3P water molecules em-ploying period boundary conditions orlarge droplets. Parameters are acceptedor rejected on the basis of agreement be-tween experimentally observed and calcu-lated structural and thermodynamic proper-ties. Applications of the results tocalcium-binding proteins, eg. calmodulin,will be presented and implications regardingthe use of lanthanide ions as surrogateprobes for Ca2+ will be discussed. This re-search was supported in part by the NIH(GM23599).


M-Posl 75DISORDER IN LYSOZYME CRYSTALS. J.B.Clarage, M.S. Clarage, D.L.D. Caspar.Rosenstiel Basic Medical Sciences ResearchCenter and Department of Physics, BrandeisUniversity, Waltham, MA 02254.

Diffuse X-ray scattering in the halosabout a crystal's Bragg reflections isdetermined by the correlations in atomicmovements. We have simulated the diffuseintensity distribution for insulin andlysozyme in terms of exponential displace-ment correlation functions. In tetragonallysozyme crystals the mean square atomicdisplacements are about twice that in themore tightly packed triclinic lattice, butthe correlations in displacement are never-theless very similar. About 90Z of thedisorder can be accounted for by internalmovements correlated with a decay distanceof about 6 A; the remaining 10% correspondsto intermolecular movements that decay in adistance the order of size of the proteinmolecule. Displacement correlations do notdecay inversely with distance as in anelastic solid, but rather exponentially,just as positional correlations in aliquid. This liquid-like disorder issimilar to the disorder we have observed intwo-dimensional colloidal crystals of poly-styrene latex spheres, where repulsiveinteractions dominate.

M-Posl 77THE STRUCTURE AND DYNAMICS OFPORCINE AND RAT INSULIN DIMERS.H. Romanowskd, D. F. Steiner, and M. W. Makinen,Department of Biochemistry and Molecular Biology,The University of Chicago, Chicago, IL 60637

A molecular dynamics (MD) study has beencarried out to evaluate structural fluctuations ofporcine and rat insulin dimers in vacuo at 293K. Therefined atomic coordinates of 2Zn porcine insulin (Aconformation) at 0.15 nm resolution served as theinitial structural model for MD simulations ofporcine insulin and also as a basis for rat insulinsafter replacement of substituted amino acid residues.The MD averaged structures calculated from thefinal 50 ps portion of each trajectory showedaveraged deviations of c' atoms of 0.33 nm for therat 1-I homodimer and 0.15 and 0.165 nm for porcineand rat I-11 insulin dimers, respectively. After 65 psof MD simulations the rat I-I dimer exhibited largestructural hinge-bending oscillations betweenmonomeric units. This was not observed in the ratII-4 dimer. The potential energy of the equilibratedrat insulin dimers at room temperature is about 250kJ/mole higher than that of the porcine insulin dimer.The differences in potential energy of the differenttypes of insulin dimers is being further investigatedby the simulated annealing technique. (Supported byNIH #GM21400 and DK13914).


M-Posl 76AVERAGE WATER STRUCTURE IN CUBIC INSULINCRYSTALS. J. Badger and D.L.D. Caspar.Rosenstiel Basic Medical Sciences ResearchCenter, Brandeis University, Waltham, MA02254.

A range of phenomena demonstrate theimportance of solvent-protein interactionsin biological processes, but little isknown about the distribution of watermolecules arouind proteins. Cubic insulincrystals provide a suitable experimentalsystem for obtaining this information sincethey contain large solvent channels. Theavailable X-ray diffraction data extend to1.7 A resolution and the protein model hasbeen refined to R=0.20. By applying novelmethods for electron density map refine-ment, analysis and graphical display, wehave mapped the average electron densitydistribution in the solvent. This mapshows that water molecules throughout thecrystal (up to five water layers from theprotein surface) are significantly moreordered than in pure liquid water. Thesolvent distribution appears to be repre-sentable by a small number of fluctuatingwater networks. Extended hydration aroundprotein molecules could account for the"hydration force" that impedes molecularassociation and for anomalies in electro-static interactions.

M-Posl 78

CALCULATION OF PROTEIN CORRELATION TIMESFROM SOLVENT-ACCSSIBI E SURFACEAREAANDHYDRATION POTENTLSArthur J. Weaver, liowId Hughes Medical Institute,Univ. of Texas Southwestem Medical Center, Dallas,TX 75235-9050

A method for calculating protein correlation times(m's) based on solvent-accessible surface area andhydration potentials (Wolfenden et al.1981. Biochemistry 20: 849-855) is presentedwhich successfully predicts 'm's extracted from13C NMR relaxation data using the model-freeapproach of Upari and Szabo (1982. J. Am. Chem.Soc. 104: 4546-4559).

It has been shown that accurate determination ofthe side chain motional parameters (S,i:*) by thismethod is dependent on 'Em values determinedexperimentally or by calculation (Weaver et al.,Biochemistry in press). However, literaturevalues for protein Em's show considerablevariation. An independent determination of 'Emeither experimentally or by calculation allows oneto test the ability of the model-free approach toextract accurate 'rm's and, by implication, S and'E*values for a given system.Supported in part by N1486K0521 from ONR.


MPosl 79

DYNAMICS OF SHORT DNA DUPLEXES: AN EPRPROBE STUDY. Eric J. Hustedt, James J. Kirchner,Andreas Spaltenstein, Paul B. Hopkins, and Bruce H.Robinson, Department of Chemistry, University ofWashingtn, Seattle, WA 98195.

We have recently developed a nitroxide spinlabel which is covalently attached to an analog ofthymidine via an acetylene linkage. The spin labeledthymidine can be site specifically incorporated intoDNA oligomers of a specific sequence using standardphosphoramidite chemistry. The acetylene linkageprovides a spacing so that the probe does not interferewith the structure of the DNA, while limiting theindependent motion of the probe.

Using this spin-probe, we have studied thedynamics of short DNA duplexes (up to 96 base pairs inlength) as a function of temperature, viscosity, andduplex length. The EPR spectrum of a spin-labelledDNA duplex is sensitive to: 1) the global tumbling ofthe DNA duplex; 2) the length-dependent collectiveinternal motions (bending and twisting) of the duplex;and 3) the independent motion of the spin-probe and/orthe thymidine to which it is attached. Simulations ofthe EPR spectra of the spin-labelled DNA duplexesenable us to quantitatively detemnine the contribution tothe lineshape from each of these three sources ofmotion. The simulations demonstrate that the globaltumbling of the duplex can be predicted accurately interms of the hydrodynamics of a rigid cylinder and thatthere is an additional length-dependent motion of thespin-probe which is due primarily 'to bending motionsof the DNA duplex.M-Posl 81

REFINEMENT OF LIPID BILAYER CRYSTALSTRUCTURES BY ENERGY MINIMIZATION. GarretVanderkooi, Chemistry Department, NorthernIllinois University, DeKalb, IL 60115.

Energy minimization calculations werecarried out on the crystal structures ofdilauryl phosphatidylethanolamine-aceticacid (DLPE-HA), palmitoyl lysophosphatidyl-ethanolamine (lysoPE), and dilauryl glycerol(DLG). The empirical energy was calculatedas the sum of intramolecular and intermolec-ular terms. Analytical first derivativesof the energy were computed with respectto bond rotations, rigid body motions,and lattice constants. Minimization wascarried out simultaneously with respectto all bond rotations (36 in DLPE-HA) andrigid body motions (12 in DLPE-HA), whilemaintaining the crystal symmetry and theexperimental lattice constants. The DLPE-HAtotal energy decreased from -130 to -143kcal/mol upon minimization,owith a meanatomic displacement of 0.21A. The lateral,head-head, and tail-tail contributionsto the intermolecular energy were -71.2,-16.8, and -2.6 kcal/mol, resp. The head-head interbilayer electrostatic forceis repulsive in DLPE if HA is omitted,but it is attractive if HA is present;for lysoPE, the head-head electrostaticforce is attractive.


M-Posl 80

EIECTROSTATIC SOLVATION FREE ENERGY: ACOMPARISON OF CONTINUUM AND FREE ENERGYPERTURBATION METHODSA. Jean-Charles, A. Tempczyk, C. Still, K. Sharp,B. Honig, Dept. of Biochemistry, ColumbiaUniversity

Solvation is important to the stability andinteractions in proteins and nucleic acids.Solvation energy can be obtained from freeenergy perturbation techniques (FEP) inwhich solute and solvent molecules are treatedexplicitly. Alternatively, the solvent can bedescribed as a continuum, retaining theexplicit solute representation. The solvationenergy can then be obtained from finitedifference solutions to the Poisson-Boltzmannequation (FDPB). A comparison of the FEP andFDPB methods for a dozen polar organicmolecules shows good agreement, e.g., forMeOH the electrostatic component of solvationis -6.22(-6.88)kcal/mole, for acetamide -11.81(-10.77), for N,N-dimethyl-acetamide -6.5(-7.45).FEP results are in parentheses. Since the FDPBresult entails only a thousandth of thecomputational cost of a FEP result, our resultssuggest that FDPB maybe a very efficient wayof obtaining electrostatic solvation energiesin proteins and nucleic acids.

M-Posl 82

DIELECTRIC CONSTANT OF PROTEINCRYSTALSH.G.L. Costera, X. Chengb and B.P. SchoenbornCenter for Structural Biology, Department ofBiology, Brookhaven National Laboratory, Upton,New York 11973

Dielectric constants are important constituentsin calculating electrostatic properties of proteincrystals. In general, an estimated interior lowdielectric constant of e =2-5, and a solvent highdielectric constant of c =40-80 are currently usedin the absence of experimental values. Thedielectric substructure of protein crystals gives riseto interfacial polarization at the boundaries ofprotein and solvent. This manifests a dispersionwith frequency of the overall capacitance andconductance of protein crystals. We will presentmeasurements of the dispersion in capacitance andconductance to characterize the dielectricparameters of the substructure of myoglobincrystals. This method uses a four terminal highprecision measuring system (a digitalspectrometer) for the determinations ofimpedance and phase angle, at very low frequency.a University of New South Wales, Sydney(Australia)b State University of New York at Stony Brook,New York

W0a Biophysical Joumal vol 57, 1990

M-Posl 83

A BIFURCATING ULTRAMETRIC DIS-TRIBUTED SYSTEM (BUDS) FOR SINGLETRYPTOPHAN PROTEIN FLUORESCENCEN. Silva & E. Gratton. UIUC, LFD, 1110 W.Green St., Urbana, IL 61801.Single tryptophan protein fluorescence dis-plays a complex be7havior, especially at lowtemperature. The fluorescence decay hasbeen described by a continuous distributionof decay rates. The physical origin of the dis-tribution has been attributed to conforma-tional substates. Previous models have beenuseful in describing fluorescence decay overa wide temperatre range. BUDS is proposedto explore ihe fluorescence behavior of sin-

gle tryptopan residue roteins. BUDS intro-uces conformations substates into the

dynamics) through a hierarchy of conforma-tional substates, and incorporates variationsin protein structure through a distributionof hierarchical activation Barriers. The keyfeature of BUDS is the non-exponentialbehavior of the fluorescence at low tempera-tures. BUDS is applied to both fluorescencelifetime and anisotropy of single tryptophanroteins. To et meaningful parametersrom the mode, fluorescence experiments ofthe protein are done over a wide tempera-ture range. Bokth liand bound and unboundproteins are investigated, as well as dena-tured forms of these proteins.(Supported by PHS-P41-RR03155)

M-Posl 85GLOBAL ANALYSIS OF DISTANCE DISTRIBUTIONDATA FOR .

DONOR-ACCEPTOR PAIRS WITHDIFFERENT FORSTER DISTANCES.W. Wlczk, P.S. Els, M.N. Fishman, J.R. Lakowlcz,University of Maryland Medical School, Department ofBiochemistry, Baltimore, Maryland, and M.L Johnson,University of Virginia, Department of Pharmacology,Chariottesvlle, Virginia.

Global analyses of frequency-domain and steady-statefluorescence energy transfer measurements on D-A pairswith differert Frster distances (Rj) were performed toykid Increased resolution In the distance distribution data.Eight compounds were synthesized with R,s ranging from6-32 A. Each compound contained a tryptamine donorconnected by a ten carbon alkyl chain to one of thirteendifferent acceptors. Both frequency-domain and steady-state energy transfer measurements were performed onthese compounds. These results were compared withene-to-end distance distributions which were predictedusing a rotational isomer model and molecular dynamicscomputer simulations. Measurement of these compoundsby steady-state and time-dependent methods should giveadditional inforation about the shape of the distancedistribution. In addition, I the same distance distributionsare recovered from these molecules, which have differentacceptors and thus different transition dipole momentdirections, then one can conclude that a K. value of 2/3Is a good approximation for Ro calculations.


M-Posl 84CATALYSIS OF EUTECTIC CRYSTALLIZATION OFSUGARS BY LIQUID-LIQUID PHASE SEPARATION ONDEEPLY FROZEN POPULUS, Allen Hirsh,American Red Cross, Rockville, MD; EricErbe, USDA, Beltsville, MD; and Thomas Bent,COMSAT Corp., Clarksburg, MD.

Winter wood from Populus balsamifera v.virginiana, balsam poplar, survives coolingto liquid nitrogen at rates <50C/hr.Analysis of the wood by differentialscanning calorimetry (DSC) and dynamicmechanical analysis (DMA) shows thatliquid-liquid phase separations occur inintracellular fluids leading to sugar-richdomains with a high glass transition,approximately -300C, and protein richdomains with a low glass transition,approximately -1100C. Cycling between 0°Cand -700C at 3°C/hr. increased injuryprogressively until total mortality wasseen after 6 cycles. DMA analysis showedthat this was concurrent with precipitationout of solution of the protective intra-cellular sugars. Cycling 6X at 3°C/hrbetween 0°C and -200C or -200C and -700Cwas not injurious. These results lead usto postulate that an unusual seeding of theeutectic phase of the intracellular sugarstakes place at very low temperatures in thesugar-dilute, protein rich phase of theintracellular milieu.Supported in part by NIH Grant GM17959.M-Posl 86DIFFUSE SCATTERING AND MOLECULARMOTIONS Or TROPOMYOSINSusan Chacko*, Frank G. Whitby and GeorgeN. Phillips, Jr.Rice University, Houston, TX 77005*University of Illinois at Urbana-Champaign.

Diffuse X-ray scattering is linked tothe inter- and intra-molecular motions oftropomyosin within crystals. Diffusescattering data along some of the majordirections of the previously solvedBailey crystal form of tropomyosin havebeen measured. Various models for thethree-dimensional motions of tropomyosinin the crystal have been tested bycomparing their predicted diffusescattering patterns with the experimentaldata. Thus the probable modes of motionhave been determined. Work is inprogress to study the perturbations inthe diffuse scattering caused by thepresence of troponin in these crystals.Structure determination and diffusescattering studies are also beingconducted on a more densely packedspermine-induced crystal form oftropomyosin.

Supported by NIH grant AM32764, WelchC-1142, NSF DMB 87-16507. F.G.W. issupported by an NIH training grant.


M-Posl 87

LOW ENERGY SOLUTION CONFORMATIONSOF PIRENZEPINE DERIVATIVES.Kari J. Seger and Leonore A. Findsen, Collegeof Pharmacy, University of Toledo, Toledo, OH43606Research on effective pharmaceuticals for the

treatment of Alzheimer's disease has recentlyfocused on the muscarinic activation of the centralnervous system. Specifically, selective post-synaptic Ml and presynaptic M2 receptor bindingcompounds are being studied. These two receptortypes are best defined by pirenzepine and AF-DX116, respectively. As experimental binding data forthese derivatives becomes available, these resultswill be applied in determining structure-activityrelationships.This study consists of calculating low energy

solution conformations of a series of stericallyconstrained pirenzepine derivatives formed by thesystematic unsaturation of the pyradine ring. Themethod used is simulated annealing with the watersexplicitly included. Calculations are performedusing the program AMBER. The changes in therelative position of the ring nitrogen as a function ofdegree of unsaturation is examined.

M-Posl 89PREDICTED THREE DIMENSIONAL STRUCTURES FORNON-CRYSTATITNE PROTEINST. F. Kumosinski, E. M. Brown,C. C. Haggerty and H. M. Farrell, Jr.USDA, ARS, Eastern Regional ResearchCenter, Philadelphia, PA 19118

Three very different classes ofnon-crystalline proteins are the collagenfamily, the milk caseins, and the serumapolipoproteins. 3D structures wereconstructed using the Sybyl-Mendylmolecular modeling programs. To minimizethe probability of obtaining a spuriousstructure, initial structures incorporatedthe results of sequence based predictionsand spectroscopically obtained estimates ofsecondary structures. All structures wererefined with the Kollman Force Field EnergyMinimization calculation. Van der Waalsand electrostatic potential maps weregenerated from the refined structures, aswell as geometric details including inter-residue distances and H-bond locations.Molecular dynamics calculations based onthe refined structures showed relativemobilities of specific residues or chains.All structure showed agreement with globalphysical and biochemical informationconcerning the proteins.


M-Posl 88

DYNAMIC LONG-RANGE ORDER IN EHETERO-GENEOUS BIOLOGICAL SYSTEMS: BOVINEMILK. P. Cooke, L. Kakalis, T. Kumosinski,H. Farrell, & M. Thompson. USDA/ARS/ERRC,Philadelphia, PA 19118.Milk is a complex liquid secreted by epithelial cells

of mammary tissue. It contains two unique supramol-ecular aggregates, casein micelles and plasma mem-brane-bounded lipid droplets suspended in a solublephase, containing whey proteins, lactose, and ions.While the properties of the micelles and lipid havebeen studied independently, little attention has beendirected at their kinetic interactions. Video-enhancedoptical phase contrast microscopy of thin films ofmilk resolves casein micelles in rapid motion inter-spersed with the lipid droplets. Spatially filteredimages reveal that the distribution of micelles is notuniform; micelles are clustered into aggregates with apersistence of 15-20 secs and fractal dimensions of1.3-1.7. The aggregates have a uniform long-rangeinterspace of 10- 15 micrometers. Skim milk andpurified casein micelles are also organized into thesefractal aggregates in patterns with similar long-rangeorder, suggesting that the lipid droplets do not playan essential structural role. These methods may beuseful for analyzing dynamic weak interactions inheterogeneous biological media.

M-Posl 90MOLECULAR DYNAMICS SIMULATIONS

OF PEA LECTIN

Gretchen Meinke (Intro. by F. L. Suddath)School of Chemistry and Biochemistry, GeorgiaInstitute of Technology, Atlanta, GA 30332

Once a three dimensional model of a protein is availablefrom experimental techniques such as X-raycrystallography, this model may serve as the startingpoint of a theoretical investigation of that protein usingthe computational intensive technique of moleculardynamics (MD). This method involves solving theequations of motion with respect to time for each atomin the protein and generates a trajectory of theprotein. One then has information about the kinds andamplitudes of motions available to that protein. Theprotein pea lectin, a dimer, has been refined in ourlaboratory to 1.7 A resolution. A lectin is a proteinwhich is characterized by its ability to agglutinatecells and precipitate complex carbohydrates . Lectinshave been isolated from a wide variety of sources, butthe best characterized lectins are those isolated fromplant seeds. Pea lectin is isolated from the commongarden pea, Pisum sativum. MD simulations arecurrently being performed on the protein pea lectinunder various conditions: in vacuo (without anysolvent molecules explicitly present), with a 4-layershell of water molecules, and ultimately in a box ofwater using periodic boundary conditions.

82a Biophysical Joumal voL 57, 199O

M-Posl 91

SIMULATION OF MACROMOLECULAR INTERACTIONSDURING SIZE-EXCLUSION CHROMATOGRAPHY:POSITIVE COOPERATIVITY CONTRIBUTION BY GELMATRIX. F. J. Stevens, Biological andMedical Research Division, ArgonneNational Laboratory, Argonne IL 60439.The interactions of ternary combinations

of antibody and antigen during size-exclu-sion chromatography are relevant in compe-tition experiments to determine relativeepitope specificity and interactionkinetics. Peptide-mediated cross-linkageof antibodies in polyclonal antisera re-presents another ternary interaction thatmay be observed chromatographically. Inthe presence of the porous sequesteringenvironment of a gel matrix, otherwisechemically and sterically independentinteractions exhibit a characteristic ofpositive cooperativity; the formation ofone antibody:epitope bond is dependent onthe concentration of the second antibody.The formation of one complex decreases thesolvent volume accessible to antigen andresults in the presentation of the secondepitope to the remaining antibody. Com-puter simulation is used to examine thisphenomenon, which may have physiologicalanalogs. This work supported by contractW-31-109-ENG-38 from the Office of Healthand Environmental Research.

MACROMOLECULAR THEORY

M-Posl 92MULTI-SCALE ISING MODEL FOR PROTEIN DENA-TURATION. Peter Leopold (Intro. by Mauricio Montal),Lab. of Bio. Dyn. & Theo. Med. and Dept. of Phys., B.019, UCSD, La Jolla, CA 92093.In order to relate protein denaturation and the underlyinggeometry of secondary structure interactions, we have nu-merically investigated an Ising-like model of protein secon-dary bondsl . A key feature of the model is its reliance onX-ray crystal coordinates for defining the elemental hydro-gen, cystine and salt bonds and hydrophobic contacts andtheir nearest neighbors. The free energy Hamiltonian H(T) --Z(ei(T)-aT)bi-JEbib. is numerically simulated forhydrated lysozyme and used to calculate the fractional fold-ing <b> (Fig. A). Here, aT and ei( are the entropic andenthalpic energies per broken bond bi(wherebi=± 1), and Jis the nearest-neighbor coupling constant. We then recast theproblem in terms of an ordered, inhomogeneous Ising sys-tem. The nearest-neighbor bond distribution is best fit by along-tailed Levy distribution (Fig. B) with exponent 8=0.8.Thus lysozyme is shown to possess a multi-scale secondarybond geometry that adequately exhibits denaturation and in-vites substructural fluctuation studies.lkegami,A.,et.al.,Biophys.Chem. 6:117-149(1977).

A Lysozyme' DelnOturation pfI-4.5 S .YSOYzMrP: LEVY 6 -.0

E(T). 30- 04 70.I/7

200 300 400 500 000 700 0 10 00304000TOIIPEOAURE {K) NUMBR 0r 120330 ogooOo

thirty-fourth annual meeting february 18-22, 1990 ...asher/homepage/spec_pdf... · invasion and...

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