evidence for effect of gm1 on opioid peptide conformation: nmr study on leucine enkephalin in...

Evidence for Effect of GM1 on Opioid Peptide Conformation: NMRStudy on Leucine Enkephalin in Ganglioside-Containing Isotropic

Phospholipid Bicelles

Anindita Gayen and Chaitali Mukhopadhyay*

Department of Chemistry, UniVersity of Calcutta, Kolkata 700 009, India

ReceiVed December 28, 2007. ReVised Manuscript ReceiVed February 25, 2008

Enkephalins are endogenous neuropeptides that have opioid-like activities and compete with morphines for thereceptor binding. The binding of these neuropeptides to membrane appears crucial since enkephalins interact withthe nerve cell membranes to achieve bioactive conformations that fit onto multiple receptor sites (µ, δ, and κ). UsingNMR spectroscopy, we have determined the solution structure of the small opiate pentapeptide leucine enkephalinin the presence of isotropic phospholipid bicelles: phosphocholine bicelles (DMPC:CHAPS 1:4) and phosphocholinebicelles doped with ganglioside GM1 (DMPC:CHAPS:GM1 1:4:0.3). Bicelles containing GM1 were found to interactstrongly with leucine enkephalin, whereas a somewhat weaker interaction was observed in the case of bicelles withoutGM1. Structure calculation from torsion angles, chemical shifts, and NOE-based distance constraints explored thatthe peptide could flexibly switch between several µ- and δ-selective conformations in both the bicelles though µ-selectiveconformations turned out to be geometrically preferred in each bicellar system. A detailed analysis of the structurespresented supports the variance over the singly associated conformation of enkephalin in nerve cell membranes.

Introduction

Enkephalins are a class of endogenous opioids that controlpain, emotion, and regulate respiratory, cardiovascular, andgastrointestinal functions.1 This group of opioids are found incentral nervous systems and the gastrointestinal tracts, wherethey bind preferentially to the δ-opiate receptors with higheraffinity than for the µ-receptors.2 There are two naturally occurringenkephalins with sequence YGGF (L or M), which differ onlyin the identity of the C-terminal amino acid. Since theiridentification in 1975,3 extensive spectroscopic studies are beingperformed on these two peptides to investigate the conformationand the mechanism of the action of the enkephalins to themembranes, which is still not well understood.4

The conformational behavior of enkephalins is acutelydependent on the solvent5 and the ionic state.6 In aqueous medium,leucine enkephalin is believed to be zwitterionic and a confor-mational equilibrium persists among the different possible statesof the peptide over a single preferred conformation.7 However,a recent analysis of Raman spectra8 corroborated with DFT(density functional theory) calculations9 and MD (moleculardynamics) simulation studies5 suggested that, leucine enkephalinhas a tendency to form a compact bent structure in water.Conversely, though it is well accepted that, in membrane medialike micelles and vesicles, leucine enkephalin adopts a well-

defined �-turn conformation,10,11 some recent studies on interac-tion of enkephalins with model membranes, like bicelles,4,12–15

have shown the conformational diversity and flexibility of thepeptide in membranes.

Previous reports stated that enkephalins are able to bind thenegatively charged lipids like phosphatidic acid, phosphati-dylserine, and lyso-phosphatidyl glycerol with high prefer-ence.16–18 We have studied before the effect of GM1 (gangliosidemonosialo type-1), an anionic representative ganglioside, onenkephalins.19 Gangliosides are a class of glycolipids that consistsof a significant fraction (5–10 mol %) of brain lipids. It is reportedthat gangliosides can regulate the opioid receptors in neurotrans-mitter cells and enkephalins affect the synthesis of gangliosidesin central cell lines.20 This report offers a correlation betweenenkephalins and the gangliosides, the mechanism of which isstill not clear. Our previous observations regarding the interac-tion of leu-enkephalin in micelles of GM1 have shown that, inthe presence of GM1, leucine enkephalin adopts a turn structurestabilized by a hydrogen bond between the N-terminal tyrosineand the C-terminal leucine.19 The Gly3 and Phe4 R-protons arein the tightest contact with the GM1 micelles, and the Tyr1 andthe Phe4 aromatic ring protons are also involved in the interaction.Leu5 side chains were found to be least involved. Still, micelles

* Corresponding author, [email protected].(1) Steiner, H.; Gerfen, C. R. Exp. Brain Res. 1998, 123, 60–76.(2) Meng, F.; Ueda, Y.; Hoversten, M. T.; Thompson, R. C.; Taylor, L.; Watson,

S. J.; Akil, H. Eur. J. Pharmacol. 1996, 311, 285–292.(3) Hughes, J.; Smith, T. W.; Kosterlitz, H. W.; Fothergill, L. A.; Morgan,

B. A.; Morris, H. R. Nature 1975, 258, 577–579.(4) Chandrasekhar, I.; Gunsteren, W. F.; Zandomeneghi, G.; Williamson,

P. T. F.; and Meiler, B. H. J. Am. Chem. Soc. 2006, 128, 159–170.(5) Spoel, V. D. S. D.; Berendsen, H. J. C. Biophys. J. 1997, 72, 2032–2041.(6) Aburi, M.; Smith, P. E. Biopolymers 2003, 64, 177–188.(7) Graham, W. H.; Carter, E. S. H.; Hicks, R. P. Biopolymers 1992, 32,

1755–1764.(8) Abdali, S.; Jensen, M. Ø; Bohr, H. J. Phys.: Condens. Matter 2003, 12,

S1853–S1860.(9) Abdali, S.; Refstrup, P.; Faurskov, N. O.; Bohr, H. Biopolymers 2003, 72,

318–328.

(10) Rudolph-Bohner, S.; Quarzago, D.; Czisch, M.; Ragnarsson, U.; Moroder,L. Biopolymers 1997, 41, 591–606.

(11) Dhanasekaran, M.; Palian, M. M.; Alves, I.; Yeomans, L.; Keyari, C. M.;Davis, P.; Bilsky, E. J.; Egleton, R. D.; Yamamura, H. I.; Jacobsen, N. E.; Tollin,G.; Hruby, V. J.; Porreca, F.; Polt, R. J. Am. Chem. Soc. 2005, 127, 5435–5448.

(12) Marcotte, I.; Dufourc, E. J.; Ouellet, M.; Auger, M. Biophys. J. 2003, 85,328–339.

(13) Marcotte, I.; Separovic, F.; Auger, M.; Gagne, S. M. Biophys. J. 2004,86, 1587–1600.

(14) Arnold, A.; Labrot, T.; Oda, R.; Dufourc, E. J. Biophys. J. 2002, 83,2667–2680.

(15) Whiles, J. A.; Brasseur, R.; Glover, K. J.; Melachini, G.; Komives, E. A.;Vold, R. P. Biophys. J. 2001, 80, 280–293.

(16) Milon, A.; Miyazawa, T.; Higashijima, T. Biochemistry 1990, 29, 65–75.(17) Marcotte, I.; Ouellet, M.; Auger, M. Chem. Phys. Lipids 2004, 127, 175–

187.(18) Nilsson, M.; Morris, G. A. Magn. Reson. Chem. 2006, 44, 655–660.(19) Chatterjee, C.; Mukhopadhyay, C. Biopolymers 2003, 70, 512–521.(20) McLawhon, R. W.; Schoon, G. W.; Dawson, G. J. Neurochem. 1983, 41,

1286–1296.

5422 Langmuir 2008, 24, 5422-5432

10.1021/la704056d CCC: $40.75 2008 American Chemical SocietyPublished on Web 04/16/2008

cannot be exact model membranes because of the small radiusof curvatures, and often micelles are found to misfold, denature,or aggregate the peptide and the protein structures.21 In the presentstudy, GM1-doped bicelles are used as the ideal model membranesfor studying the effect of GM1 as a natural nerve cell membraneconstituent on the conformation of the leucine enkephalin.

Several recent publications have reported on the preparation,characterization, and applications of bicelles.22,23 Bicelles gener-ally consist of long- and short-chain phospholipids, where thesize and shape are controlled by the ratio (q) of the long chainphospholipids to the shorter ones. At higher q values (q > 2.5),bicelles adopt a magnetically aligned lamellar bilayer morphologywith ellipsoidal shape.24 In contrast, at smaller q values (q < 1),bicelles are known to have fast tumbling and isotropic propertiesin aqueous solution and maintain discoidal shape.13 Isotropicbicelles permit high-resolution NMR measurements of membrane-bound molecules and therefore can potentially serve as suitablesystems for structure determination of small membrane-associatedpeptides, which relates to their biological activities. 21 However,it is also necessary to gain insight into the peptide-membraneinteraction to elucidate the location, orientation, and dynamicsof the peptides on the membrane, and for this purpose, the solid-state and the solution-state NMR experiments using the magneti-cally oriented bicelles are applied very successfully.24–26

In this study, we have used fast tumbling isotropic bicelles (q) 0.25) (justified in Supporting Information) comprised of (1)1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 3-(chola-midopropyl)-dimethylammonio-2-hydroxyl-1-propane-sul-fonate (CHAPS) and (2) DMPC, CHAPS, and GM1 (structuresof DMPC, CHAPS, and GM1 are given as the SupportingInformation, Figure S1) to determine the membrane-associatedstructure of the small opiate peptide, leucine enkephalin (LENK).GM1 was incorporated in the bicelles at 30 mol % of thephospholipid DMPC. This particular ratio of GM1 (GM1 containsmonosialic acid) to PC was used to make the bicelle anionic.13

Moreover, DMPC:CHAPS:GM1 in ratio 1:4:0.3 represents abicelle that contains 5.66 mol % GM1, so the nerve cell membranecomposition (5–10 mol % GM1) is also well maintained.27

CHAPS, the cholesterol mimic, was used because cholesterol isa true membrane constituent28 and CHAPS offers a similarrigidifying effect to the membrane as cholesterol.29,30 1H and 13Cone-dimensional and two-dimensional NMR along with fluo-rescence spectroscopy were used as the conventional methodsfor the peptide-structure investigation. We have found that theinteraction of the GM1 containing bicelles is stronger with thepeptide LENK than the zwitterionic phosphocholine bicelles,which provides the greater utility of the GM1-doped isotropicbicelles. Finally, our results show that leucine enkephalin canadopt several µ- and δ-selective conformations in membrane

environments, but the µ-selectivity is preferred over theδ-selectivity in each bicellar system. Thus we have discussed thebiological relevance of conformers of leucine enkephalin in modelmembranes that links to their receptor subtype selectivity.31

Experimental SectionMaterials. Protonated DMPC and CHAPS were obtained from

Sigma-Aldrich and used without further purification. GM1 wasisolated and purified from goat brain in laboratory following thepublished protocol.19 Leucine enkephalin was purchased from Sigma-Aldrich and was used without further purification. Deionized waterwas used in the experiments because salt interference was observedon the binding of enkephalins to phosphocholine vesicles.32

Bicelle Preparation. To prepare the bicelle, first a 75 mM stocksolution of DMPC was prepared by suspending 5.084 mg of DMPCin 100 µL of H2O. The solution was vortexed and centrifuged atroom temperature, and the pellets of DMPC were resuspended withvortexing. When the cycle was repeated for at least 10 times, auniform homogeneous dispersion of DMPC was obtained. NowCHAPS from a 500 mM stock solution (in water) was added to theDMPC dispersion to achieve q ) 0.25. The sample was vortexeduntil the solution was clear and transparent. The solution was againcentrifuged, and no pelleted lipid was separated from the sampleafter the centrifugation step. GM1 was incorporated in the bicellesat 30 mol % of the phospholipid (DMPC) to prepare bicelles withGM1. GM1 and DMPC were weighed first to prepare a homogeneoussolution of GM1 and DMPC, and then CHAPS was added inappropriate amounts to make the solution clear following the protocolas stated above. The final dilution was done by deionized water ofpH 6.6 to make cL (concentration of lipid) ) 4% w/v of each bicellesolution. The pH was adjusted by adding small volumes of 1 M HClor 1 M NaOH. No buffer was used in order to keep the ionic strengthminimum.33 For the PC bicelle, the molar ratio of DMPC:CHAPSwas 1:4 and for the GM1 containing bicelle, the molar ratio amongthe constituents was DMPC:CHAPS:GM1 1:4:0.3. Prior to the NMRmeasurements with peptide, the quality and character of the GM1containing bicelles were examined using 31P NMR experiment andTEM (transmission electron microscopy) and compared with thewell-characterized bicellar mixture of DMPC/CHAPS (experimentaldetails along with the representative NMR spectra and electronmicrographs are described in Supporting Information, Figures S3and S4) to see whether the bicellar morphology was maintained inthe presence of GM1 (the critical micelle concentration (cmc) wasin the order of 3 × 10-8 to 3 × 10-6 M).34 It was found that withsome differences the small bicelle morphology (see SupportingInformation) is mostly maintained in the presence of GM1.35–38

Therefore, GM1 does not act as a detergent destroying bicellarstructure thus confirming the applicability of the GM1-doped bicellesto the following structural study of leucine enkephalin. Leucineenkephalin solid was added to the ready-made bicelles (DMPC/CHAPS 1:4 and DMPC/CHAPS/GM1 1:4:0.3 molar ratio) in thedesired peptide:lipid ratio (lipid/peptide 13:1) for the experiments,4

and then the solution was again vortexed and then stored at -20 °Cbefore the experiments. At-20 °C, without a peptide LENK (leucineenkephalin), the prepared stock bicelle samples were observed toremain stable over months.

Fluorescence Spectroscopy. The fluorescence experiments weredone using a Perkin-Elmer LS-50B spectrometer at the room

(21) Poget, S. F.; Cahill, S. M.; Girvin, M. E. J. Am. Chem. Soc. 2007, 129,2432–2433.

(22) Cardon, T. B.; Dave, P. C.; Lorigan, G. A. Langmuir 2005, 21, 4291–4298.

(23) Minto, R. E.; Adhikari, P. R.; Lorigan, G. A. Chem. Phys. Lipids 2004,132, 55–64.

(24) Dürr, U. H. N.; Yamamoto, K.; Im, S.-C.; Waskell, L.; Ramamoorthy,A. J. Am. Chem. Soc. 2007, 129, 6670–6671.

(25) Dvinskikh, S. V.; Dürr, U. H. N.; Yamamoto, K.; Ramamoorthy, A.J. Am. Chem. Soc. 2007, 129, 794–802.

(26) Dvinskikh, S. V.; Dürr, U. H. N.; Yamamoto, K.; Ramamoorthy, A. J.Magn. Reson. 2007, 184, 240–247.

(27) Chi, E. Y.; Frey, S. L.; Lee, K. Y. C. Biochemistry 2007, 46, 1913–1924.(28) Ahmed, S. N.; Brown, D. A.; London, E. Biochemistry 1997, 36, 10944–

10953.(29) Andersson, A.; Biverstahl, H.; Nordin, J.; Danielsson, J.; Lindahl, E.;

Maler, L. Biochim. Biophys. Acta 2007, 1768, 115–121.(30) McKibbin, C.; Farmer, N. A.; Jeans, C.; Reeves, P. J.; Khorana, H. G.;

Wallace, B. A.; Edwards, P. C.; Villa, C.; Booth, P. J. J. Mol. Biol. 2007, 374,1319–1332.

(31) Schwyzer, R. Biochemistry 1986, 25, 6335–6342.(32) Milon, A.; Miyazawa, T.; Higashijima, T. Biochemistry 1990, 29, 65–75.(33) Auge, S.; Bersch, B.; Tropis, M.; Milon, A. Biopolymers 2000, 54, 297–

306.(34) Sonnino, S.; Cantu, L.; Corti, M.; ACquotti, D.; Venerando, B. Chem.

Phys. Lipids 1994, 71, 21–45.(35) Matsumori, N.; Morooka, A.; Murata, M. J. Am. Chem. Soc. 2007, 129,

14989–14995.(36) Richard, J.-A.; Kelly, I.; Marion, D.; Pezolet, M.; Auger, M. Biophys. J.

2002, 83, 2074–2083.(37) Glover, K. J.; Whiles, J. A.; Wu, G.; Yu, N.; Deems, R.; Struppe, J. O.;

Stark, R. E.; Komives, E. A.; Vold, R. R. Biophys. J. 2001, 81, 2163–2171.(38) Hirotaka, S.; Fukuzawa, S.; Kikuchi, J.; Yokoyama, S.; Hirota, H.;

Tachibana, K. Langmuir 2003, 19, 9841–9844.

Effect of GM1 on Leucine Enkephalin Conformation Langmuir, Vol. 24, No. 10, 2008 5423

temperature. An excitation wavelength of 275 nm was used for allthe experiments. Background intensities of the PC (phosphocholine)bicelle and the GM1 containing PC bicelle without LENK weresubtracted from each LENK-bicelle spectrum to discard anycontribution from the solvent. The concentration of the peptide insolution was 50 µM, and small aliquots from the stock solution ofthe bicelles were added gradually to the peptide solution so that theconcentration of the peptide remained unaltered in the medium. Thesame fluorescence experiments were repeated with 3.5 M K2HPO4

present in the solution.NMR Spectroscopy. All homonuclear NMR experiments were

performed on a Bruker DRX 500 MHz spectrometer equipped witha 5 mm broadband inverse probe head. Experiments were carriedout at 25 °C, and referenced to the 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid, sodium salt (TSP). The heteronuclear NMRexperiments were performed on a Bruker AVANCE 500 MHzspectrometer equipped with a 5 mm broadband observe probe head.Suppression of the water signal was typically accomplished bygradient methods of suppression using WATERGATE.

High-resolution one-dimensional 1H NMR spectra were acquiredfor the bicelles and peptide/bicelle mixtures in aqueous solution(90% H2O, 10% D2O). Two-dimensional TOCSY (total correlationspectroscopy), DQF-COSY (double quantum filtered correlationspectroscopy), ROESY (rotating Overhauser effect spectroscopy),and NOESY (nuclear Overhauser effect spectroscopy) experimentswere collected in the phase sensitive mode. All the spectra werereferenced to the HDO peak at 4.709 ppm. The TOCSY pulsesequence included 80 ms MLEV-spin lock, and the ROESY andNOESY spectra were acquired with a mixing time of 300 ms. Thespectral width was 6 kHz in both direct (F2) and indirect (F1)dimensions, with 2048 complex data points in F2 and 512 complexdata points in F1. Typically, 16 scans were taken per increment. TheHSQC (heteronuclear single quantum coherence) experiment wasrecorded with 1024 data points in the F2 (13C) dimension and 256data points in the F1 (1H) dimension with 48 scans per incrementin both dimensions. A spectral width of 5482.45 Hz was set in theF2 dimension. The spectra were processed using XWINNMR version3.75 and analyzed using SPARKY version 3.112.

1H resonances in the TOCSY spectra were assigned using thestandard sequential assignment procedure. Each of the cross-peaksin the ROESY and the NOESY spectra was integrated, and thevolumes were converted to distance restraints. Cross peaks werecategorized13 as strong, medium, weak, and very weak based ontheir intensities. The conservative upper distances were fixedrespectively as 3.5, 4.0, 4.5, and 6.0 Å with a lower distance limitof 1.8 Å. The upper distance limits were normalized against theknown distance of 3.05 Å for the Phe4

HN-HRNOE (nuclear Overhausereffect) for the nonaromatic proton NOEs and the 2.48 Å for theTyr1

Hδ-Hεfor the aromatic proton NOEs. Here, corrections of 0.5 Åwere applied to the upper bound distances derived from NOEs toaccount for the spin diffusion effects. The dihedral angles werecalculated from the 3JHN-HR coupling constants measured from theDQF-COSY spectra using the Karplus relation A cos2 φ + B cosφ + C ) 3JHN-HR, where A ) 6.98, B ) -1.38, and C ) 1.72 with-60° as the phase difference in the dihedral φ. RC chemical shiftswere determined using the RC resonances identified by directinvestigation of the RH-RC regions of the HSQC spectrum.

Structure Calculations. Distance restraints were obtained fromthe combination of NOESY and ROESY experiments (τmix ) 300ms) for enkephalin in water, PC, and GM1 containing PC bicelles.Backbone torsion angle constants extracted from DQF-COSY wereused for two residues Phe4 and Leu5.13 Two hundred and fiftystructures were generated from the random starting conformationusing the standard simulated annealing protocol of XPLOR-NIH.39

The highest temperature that was achieved during the SA (simulatedannealing) protocol was 3000 K, and the final lowest temperaturethat was achieved, was 12.5 K. A repel constant of 1.2 was used.The top structures from the SA were further refined using the

refinement protocol of XPLOR-NIH. Several rounds of structurecalculations were carried out, and depending on the NOE violations,the distance constraints were adjusted. PROCHECK-NMR40 wasused to check the quality of the structures. Structural figures werevisualized and analyzed using VMD-XPLOR software packageversion 1.5 Linux 2.4_i686 running on Linux41 and RasMol version2.7.3.1 running on Windows XP. Software MOLMOL (ETH, Zurich,Switzerland) was used to illustrate the Ramachandran plots.42

Determination of the distances between the centers of the aromaticrings, the side chain �1 and �2 angles, and the calculation of theH-bonds was done using Insight II software (98.0 version, AccelrysInc.) running on a Silicon Graphics O2 workstation.

The resonance assignments, coupling costants, and constraintswere submitted to BioMagResBank under accession number 20003for leucine enkephalin in water, DMPC/CHAPS bicelles, and DMPC/CHAPS/GM1 bicelles and the corresponding ensembles of 90structures were deposited to SMSDep (PDB bank for small molecules)with RCSB ID codes 1LNW, 1LND, and 1LNG.

(39) Schwieters, C. D.; Kuszewski, J. J.; Tjandra, N.; Clore, G. M. J. Magn.Reson. 2003, 160, 65–73.

(40) Laskowski, R. A.; Rullmannn, J. A.; MacArthur, M. W.; Captein, R.;Thornton, J. M. J. Biomol. NMR 1996, 8, 477–486.

(41) Schwieters, C. D.; Clore, G. M. J. Magn. Reson. 2001, 149, 239–244.(42) Koradi, R.; Billeter, M.; Wuthrich, K. J. Mol. Graphics 1996, 14, 51–55.

Figure 1. Fluorescence titration of (A) leucine enkephalin with DMPC/CHAPS bicelles (B) with DMPC/CHAPS/GM1 bicelle in the presenceof 3.5 M K2HPO4. The bicellar samples were prepared in H2O/D2O (9:1)(pH 6.6) and contain 4% (w/w) of phospholipids with q ) 0.25 andlipid/peptide 13:1. The lower-most spectrum is of free enkephalin, andthen successive spectra were obtained upon addition of bicelles to thepeptide in the mole ratio shown in the figure.

5424 Langmuir, Vol. 24, No. 10, 2008 Gayen and Mukhopadhyay

Results

Association of Leucine Enkephalin with the Bicelles.Fluorescence Spectroscopy. The fluorescence experiments withleucine enkephalin in bicelles were done in the presence of 3.5M potassium hydrogen phosphate, which is known to favor thetyrosinate form of the tyrosine.43 Before addition of the bicelle,the fluorescence emission spectrum of LENK (Figure 1) showsonly the peak of tyrosinate at 345 nm. With increasing amountof PC bicelle in the solution, the intensity of the tyrosine moietygradually increases and the peak shifts from tyrosinate (345 nm)to the peak of tyrosine at 312 nm. This effect is more pronouncedfor the GM1 containing PC bicelle, where the peak at 345 nmdisappears and the peak at 311 nm appears instantly upon thebicelle addition to the peptide solution indicating spontaneouschange of tyrosinate to tyrosine. The conversion of the tyrosinateto tyrosine obtained for the peptide in the GM1 containing PCbicelle is similar to the result reported earlier for leu-enkephalinin presence of GM1 micelle.19

Chemical Shift. Initially, one-dimensional 1H and 13C NMRspectra were acquired for the phospholipid bicelles and for thepeptide in the phospholipid bicelles. The 1H and 13C chemicalshifts of LENK in water and in both the bicelles are listed inTable 1. The peptide proton chemical shifts are similar (within(0.01 ppm) in both the PC and the GM1 containing PC bicelle.The one-dimensional NMR spectra of the alpha region of thepeptide in both the bicelles are presented in spectra B and C ofFigure 2 and are compared to the spectrum obtained in water(Figure 2A). The spectra for the bicellar systems are well-resolvedthat demonstrate that the low-viscosity isotropic bicellar solutionis a suitable medium for high-resolution NMR experiments.44

The comparison of spectrum A in Figure 2 with spectra B andC in Figure 2 reveals a very small change in the chemical shiftswith significant signal broadening upon binding of the peptideto both the bicelles. The Phe4H�1/H�2 and Phe4 Hδ1/H�/Hε wereresolved. But Tyr1 Hδ1/Hδ2, Hε1/Hε2, H�1/H�2, Phe4 Hδ1/Hδ2,Hε1/Hε2, Leu5 H�/Hγ, Hδ1/Hδ2 were not resolved. However, theLeu5 Hδ1/Hδ2 were resolved in the 1H-13C HSQC spectrum. Theamide protons of Gly3

HN and Phe4HN overlap in water but are

separated in the bicelles with slight upfield shift of Gly3HN asshown clearly in the amide region of the DQF-COSY spectra

(Figure 3B and Figure 3C) of LENK in the PC and the GM1/PCbicelles, when compared to that in water shown in Figure 3A.The changes in the chemical shifts (∆δ) were measured directlyfrom the 1H-1H TOCSY and the 1H-13C HSQC and therespective ∆δ values are listed in Figure 4.

NOE Assignments of Leu Enkephalin in Water, PC Bicelles,and GM1/PC Bicelles. Structures of LENK in water and in boththe DMPC/CHAPS and DMPC/CHAPS/GM1 bicelles were

(43) Okiyasu, S.; Imakubo, K. Photochem. Photobiol. 1977, 26, 541–543.(44) Vold, R. R.; Prosse, R. S.; Deese, A. J. J. Biomol. NMR 1997, 9, 329–335.

Table 1. 1H and 13C Resonance Assignments from TOCSY and HSQC Experiments

chemical shift (ppm)

residue HN HCR HC� others 3NHCR

A. 1H and 13C Resonance Assignments for LENK in Water of pH 6.6 at 25 °CTyr1 4.233 (54.14) 3.123 (36.55) 2,6 H 7.15; 3,5 H 6.86 (130.809, 115.792)Gly2 8.57 3.84, 3.91 (42.43)Gly3 7.988 3.83, 3.92 (42.133)Phe4 7.988 4.65 (54.79) 2.97, 3.18 (36.95) 2,6 H 7.266; 3,5 H 7.293; 4H 7.332 (129.387, 128.971, 128.547) 8Leu5 7.901 4.173 (54.78) 1.575 (40.875) γH 1.575; δH1 0.8439; δH2 0.8704 (24.528, 21.05, 22.38) 9

B. 1H and 13C Resonance Assignments for LENK in DMPC/CHAPS (q ) 0.25)Bicelles of pH 6.6 at 25 °C

Tyr1 4.228 (54.8) 3.125 (37.11) 2,6 H 7.153; 3,5 H 6.851 (130.83, 115.85)Gly2 8.575 3.84, 3.90 (42.47)Gly3 7.979 3.83, 3.89 (42.19)Phe4 8.001 4.64 (54.88) 2.97, 3.171 (37.27) 2,6 H 7.245; 3,5 H 7.334; 4H 7.284 (129.342, 129.071, 128.714) 8Leu5 7.869 4.17 (55.02) 1.545 (41.27) γH 1.54; δH1 0.8419; δH2 0.8664 (24.555, 21.06, 22.36) 9

C. 1H and 13C Resonance Assignments for LENK in DMPC/CHAPS/GM1 (q ) 0.25)Bicelles of pH 6.6 at 25 °C

Tyr1 4.212 (54.83) 3.11 (36.11) 2,6 H 7.137; 3,5 H 6.851 (130.9, 115.93)Gly2 8.550 3.83, 3.906 (42.69)Gly3 7.964 3.814, 3.902 (42.49)Phe4 7.989 4.642 (54.95) 2.962, 3.173 (36.982) 2,6 H 7.245; 3,5 H 7.33; 4H 7.28 (129.3, 128.7, 128.743) 8Leu5 7.875 4.157 (54.04) 1.535 (40.93) γH 1.535; δH1 0.8336; δH2 0.8659 (24.55, 21.10, 22.36) 9

Figure 2. Selected alpha regions of the 1H NMR spectra of leucineenkephalin in water (A), in DMPC/CHAPS bicelles (B), and in GM1containing DMPC/CHAPS bicelles (C). Spectra were acquired with a500 MHz NMR spectrometer at 25 °C. The bicellar samples were preparedin H2O/D2O (9:1) (pH 6.6) and contain 4% (w/w) of phospholipids withq ) 0.25 and lipid/peptide 13:1. The peptide peaks in the alpha regionin the presence of the bicelles are shown by arrows.


determined using the NOE restraints obtained from the combina-tion of 1H-1H NOESY and ROESY spectra. The amide-alpharegion of TOCSY in water is presented in Figure 5. All otherNMR spectra containing the amide-alpha and amide-amideregions of ROESY in water and in both the bicelles for LENKare given as Supporting Information, Figures S5-S10. Despitethe overlap of some peptide and lipid resonances, unambiguousROEs that were assigned lead to a total of 28, 34, and 38 restraintsfor the conformation of LENK in water, PC bicelle, and GM1/PC bicelle, respectively. The cross peaks were mostly intraresidue,or sequential. The number of cross peaks got increased when thepeptide was studied in the PC and GM1 containing PC bicelles.

No long-range cross peak signal was obtained for LENK in water,while ROE between Tyr1C�H and Phe4HN was observed inboth the bicelles. Many ROEs and NOEs were found similar inboth the bicelles, which correspond to the structural similarityamong the conformers. Cross peaks were found between Phe4HNand Leu5C�/γH and in between Phe4HN and Leu5CδH in thezwitterionic PC bicelles, which are absent in the GM1 containingbicelles. In addition, ROEs between Gly2CRH and Phe4C�H,between Tyr1C�H and Gly2CRH, Gly3CRH, and between Phe4CδHand Leu5C�/γH were only observed in the GM1 containing bicelles.However, the cross peak between Tyr1C�H and Gly2HN wasabsent in the NOESY (spectra not shown) and ROESY of LENKin GM1 containing PC bicelles. The 3JNHCR coupling constants(extracted from DQF-COSY) from which the dihedral angleswere restrained for Phe4and Leu5 were similar for both the bicelles.But, the 1H and 13C chemical shifts (Table 1), which were alsoused as additional restraints in the structure calculation, wereslightly different for the two bicelles.

Structure of Leu-Enkephalin in Water. Figure 6A presents90 structures with lowest energy out of 125 best structurescalculated for LENK in water. The structures can be divided intothree groups of conformers according to the peptide backboneand orientation of the Phe4 ring with respect to the Tyr1 ring.Group I includes 36%, and groups II and III include 33% and31% of the conformers, respectively. The statistics obtained forthe conformers in water are presented in Table 2, which showsthat the conformers have sufficient low energy with minimumviolations. The structural superposition of conformers led toaverage backbone rmsd (root-mean-square deviation) of 1.226Å. Higher average rmsd of 3.0 Å calculated for heavy atoms canbe explained by the high degree of freedom of the side chains.Individually, the overlay of conformers within groups results inaverage backbone rmsd ranging from 0.99 to 1.116 Å, whereas theheavy atom rmsd ranges between 2.427 and 2.721 Å.

The φ and ψ angles of the residues are found in the allowedregions of the Ramachandran plot (Figure 7A). The dihedralangles for the bulky residues except the N-terminal Tyr1 adoptsimilar skewed or staggered conformations in all the three groups,whereas the dihedrals of glycine residues show much fluctuation.All the structures of group I are single bent, structures of groupII are double bent, whereas conformers of group III representmostly extended structures (Figure 6A). The average distancebetween the two ends ree, the average distance between the alphacarbons of two aromatic residues C1

R-C4R,, and the average

distance between Gly2R-Leu5

R for the groups are listed in Table3 to further characterize the three groups of conformers as bent,turn, or extended.

The difference between the groups of conformers arises dueto the different orientations of the Tyr1 and Phe4 rings with respectto each other. In the structures of group I, the Tyr1 and Phe4 ringsare in the opposite side of the backbone and point toward theopposite direction. In conformers of group II, the Tyr1 and thePhe4 rings are in the same side of the backbone and point towardthe same direction.

Structure of Leu-Enkephalin in DMPC/CHAPS Bicelles.Ninety structures for LENK in PC bicelles are shown in Figure6B. They are also classified in three groups of conformersaccording to the backbone conformation and the orientation ofthe aromatic rings. To alleviate the possibility of confusion withconformers of LENK in water, these three groups are calledgroups IV, V, and VI. Group IV includes 81% of the conformerswith average backbone rmsd of 1.077 Å. The remaining 19% ofthe conformers comprising groups V (7%) and VI (12%) produceaverage backbone rmsd’s 0.711 and 1.101 Å. The heavy atom

Figure 3. Selected amide region of the 1H-1H DQF-COSY spectrumof leucine enkephalin (A) in water, (B) in DMPC/CHAPS bicelles, and(C) in GM1 containing DMPC/CHAPS bicelles. Spectra were acquiredwith a 500 MHz NMR spectrometer at 25 °C. The bicellar samples wereprepared in H2O/D2O (9:1) (pH 6.6) and contain 4% (w/w) ofphospholipids with q ) 0.25 and lipid/peptide 13:1. The Gly3, Phe4, andLeu5 residues are labeled in the figures.


rmsd’s are usually higher in all the conformers when comparedwith the backbone rmsd’s. The heavy atom rmsd’s are 2.359,1.286, and 2.828 Å for groups IV, V, and VI, respectively. Theconformational energies of the structures are low and similar tothose obtained in water (Table 2).

The backbone dihedrals are located in the more allowed regionsof the Ramachandran plot compared to the dihedrals in water(Figure 7B). Groups IV and V adopt bent conformation, whereasgroup VI shows a double bend conformation. The CR atoms ofthe tyrosine and the phenylalanine residues are ∼9 Å apart ingroups IV and VI conformers and are ∼7 Å apart in group Vconformers. The average distance between the Gly2CR and Leu5CRis ∼6 Å for the structures in group IV and is ∼8.5 Å and ∼6.5Å for the conformers in group V and group VI, respectively. Theend to end average distances (re-e) for the three groups are between9 and 12 Å as listed in Table 3. The conformers of IV and VIhave the Tyr1 and Phe4 rings located in the opposite sides of thebackbone facing toward the opposite directions and the con-formers of group V have the aromatic rings present in the sameside of the backbone facing toward each other. The Tyr1 �1 anglesare different for the three groups of conformers, whereas, thePhe4�1 angles were found nearly similar for all three groups ofstructures (Table 4).

Structure of Leu-Enkephalin in GM1 (5.66 mol %)Containing DMPC/CHAPS Bicelles. Ninety structures forLENK in the GM1 containing PC bicelles are presented in Figure

6C. These structures can be divided in mainly four groups ofconformers VII, VIII, IX, and X, where group IX can be againsubdivided in four subgroups of conformers namely IXA, IXB,IXC, and IXD. The IX conformers differ in the orientation ofthe tyrosine and the phenylalanine side chains in space. GroupsVII, VIII, and X include 52%, 14%, and 17% of the total structures,respectively. The different subgroups of group IX present a totalof 17% structures.

Figure 4. Deviations of the 1H chemical shifts ∆δ (ppm) due to binding of leucine enkephalin from bulk water to the bicellar surface (A) inthe DMPC/CHAPS bicelle and (B) in the GM1 containing DMPC/CHAPS bicelle and deviations of the 13C chemical shifs ∆δ due to binding ofleucine enkephalin from bulk water to the bicellar surface (C) in the DMPC/CHAPS bicelle and (D) in the GM1 containing DMPC/CHAPS bicelle.The bicellar samples were prepared in H2O/D2O (9:1) (pH 6.6) and contain 4% (w/w) of phospholipids with q ) 0.25 and lipid/peptide 13:1. Thecorresponding protons and carbons are indicated in the figures.

Figure 5. Selected amide region of the 1H-1H TOCSY spectrum ofleucine enkephalin in H2O/D2O (9:1) (pH 6.6). The spectrum was acquiredwith a 500 MHz NMR spectrometer at 25 °C. Cross peaks to thecorresponding protons of backbone and side chain of LENK are labeledin the figure.


The overlay of groups VII, VIII, IX, and X produces the averagebackbone rmsd of 1.215 Å and average heavy atom rmsd of3.033 Å. Group VII conformers present an average backbone

rmsd of 1.019 Å with heavy atom rmsd 2.614 Å. Group VIII andgroup X conformers produce the average backbone rmsd’s of1.028 and 0.953 Å, respectively. The subgroups of group IX

Figure 6. Backbone superimposition of the solution structures of leucine enkephalin in (A) water, (B) DMPC/CHAPS bicelles, and (C) GM1containing DMPC/CHAPS bicelles in different groups. Samples were prepared in H2O/D2O (9:1) (pH 6.6) and the bicelle samples contain 4% (w/w)of phospholipids with q ) 0.25 and lipid/peptide 13:1. All structural figures in line representation were generated by the software package VMD-XPLOR version 1.5 Linux 2.4_i686 and in ribbon representation were generated by the software RasMol version 2.7.3.1 Windows XP.


show an average backbone rmsd between 0.5 and 0.9 Å. Theheavy atom rmsd’s are again higher for all the conformers. Thebackbone dihedrals for the structures in four groups lie inthe allowed regions of the Ramachandran plot as shown in Figure7C. Table 2 represents statistics of the low energy structures,where the violations are found to be minimal.

Group VII represents a bent conformation, whereas groupVIII represents a double-bend-like conformation. The conformersof group IX show different double bent conformations withalteration in positions of Tyr1 and Phe4. Group X structures areneither bent nor turn; they are extended in nature. The averageGly2

R-Leu5R distance is ∼5.5 Å for group VII, and for the rest

the average Gly2R-Leu5

R distance is ∼8.5 Å. The end to enddistances are ∼11 Å except group VII, where the average distancebetween N- and C-termini is 7 Å. The CR atoms of the Tyr1 andPhe4 are in average 7–9 Å apart in all the conformers. The averageGly2

R-Leu5R, re-e, and the C1

R-C4R distances for all the groups

and subgroups of conformers are listed in Table 3.In the structures of group VII, the Tyr1 and the Phe4 rings are

on opposite sides of the backbone and face in opposite directions.The contrary is observed for group VIII, where, the rings are onthe same side of the backbone and face toward the same direction.In the subgroups of group IX, the aromatic rings are on the sameside of the backbone but facing the opposite direction. Thesubgroup IXB differs only by the orientation of the tyrosine sidechain �1 angle with respect to the phenylalanine side chain fromothers. The position of Phe4 is almost similar in the IXA, IXB,IXC, and IXD subgroup of conformers of group IX. The �1

angles for Tyr1 and Phe4 are listed in Table 4.Comparison of Leu-Enkephalin Structures in DMPC/

CHAPS Bicelles and DMPC/CHAPS/GM1 Bicelles. Theconformers of LENK in PC bicelles were mostly (81%) singlebent and double bent (19%). Conversely, in the GM1/PC bicelles,52% of structures were bent and 31% of structures were doublebent. No extended structure was found for the peptide in the PCbicelles, whereas in GM1 containing PC bicelles 17% of theconformers were extended. Interestingly, the structures of LENKin groups I, IV, and VII are similar. The conformers of these

Table 2. Experimental Restraints and Structural Statistics for90 Lowest Energy Structures of LENK in Water, DMPC/

CHAPS Bicelles, and DMPC/CHAPS/GM1 Bicelles (pH 6.6) at25 °C

water PC bicellePC/GM1bicelle

no. of experimental restraintsdistance of restraints from NOE

intraresidue 22 16 19interresidue 6 18 19

dihedral restraints 2 2 2XPLOR energies (kcal/mol)

Etotal -13.00 -17.86 -18.06Enoe 0.85 0.02 0.03Ebond 0.20 0.16 0.17Eangle 1.93 1.82 1.92Ecdih 0.00 0.00 0.00Eimp 0.11 0.13 0.10Erama -16.70 -20.55 -20.79Evdw 0.62 0.53 0.50

distribution of residues inRamachandran plot (%)a

most favored regions 94.5 93.4 97.8additionally allowed regions 5.5 6.6 2.2generously allowed regions 0.0 0.0 0.0disallowed regions 0.0 0.0 0.0a The program PROCHECK-NMR was used to assess the quality of

structures.

Figure 7. Ramachandran plots of the 90 lowest energy structures ofleucine enkephalin (A) in water, (B) in DMPC/CHAPS bicelle, and (C)in GM1 containing DMPC/CHAPS bicelle. The figures were generatedby the software MOLMOL. Samples were prepared in H2O/D2O (9:1)(pH 6.6), and the bicelle samples contain 4% (w/w) of phospholipidswith q ) 0.25 and lipid/peptide 13:1. Residues 4Phe (×) and 2Gly ( · )and 3Gly ( · ) are indicated in blue.


three groups are single bent with the two aromatic rings pointedin the opposite direction. No H-bond exists in any of theconformers. The differences in these structures having similarbackbone orientation can be attributed to the different averagedistances (re-e, rGly2R-Leu5R, and rC1R-C4R) (Table 3) and differentside chain �1 angles (Tyr1 and Phe4) (Table 4). The structurespresented here had no NOE violations>0.5 Å, no bond violations>0.05 Å, and no angle violation >5°.

Discussion

Association of the Peptide with the Bicelles. The aim of thepresent spectroscopic investigation is to study the interactionbetween the opioid peptides and the biological membranecontaining cholesterol and GM1. The primary informationabout the location of the peptide at the surface of the bicelleswas obtained from the fluorescence spectroscopy. In thepresence of bicelles, the peak of tyrosinate at 345 nm graduallyshifts to the peak of tyrosine at 312 nm (for PC bicelle) or311 nm (for GM1 bicelle) (Figure 1). This conversion oftyrosinate to tyrosine is found more spontaneous in presence ofthe GM1 containing PC bicelle. Hence, the interaction betweenthe peptide N-terminal and the membrane is stronger, when GM1is present in membranes. 1H NMR spectra of the peptide in the

PC (Figure 2B) and the GM1 containing PC bicelles (Figure 2C)are different, when compared to the 1H NMR spectrum in water(Figure 2A). The binding is evidenced by the changes in thechemical shifts and the signal broadening. The bicelles are foundto have an upfield effect on the Gly3HN amide proton resonance,when compared to the overlapped Gly3HN amide protonresonance with Phe4HN amide proton resonance in water (Figure3). The marked upfield shift of HR and the downfield shift ofcorresponding CR for the N terminal Tyr1 of LENK in the GM1containing PC bicelle compared to the PC bicelle suggests thatthe N terminus NH3

+ is more attracted to the headgroups at theGM1 membrane surface where sialic acid is present along withPO4

-.45 Similar observations of small chemical shifts of the 1Hand 13C signals in the N-terminal Tyr1 have been reported earlierfor methionine enkephalin on the PC-membrane surface.45 Thecarbon of the LENK C terminal CO2

– shows similar upfield shiftin both the bicelles compared to the chemical shift in water,which indicates similar interactions of the C-terminal with theamide proton of CHAPS of the bicelles. However, the Leu5CRshows a noticeable upfield shift in the presence of the GM1containing PC bicelles indicating that GM1 enhances theinteractions between the peptide and the membrane. The orderingof all HRs of LENK in the GM1 containing bicelles suggests theprominence of structure in the conformers of the peptide on theGM1-PC membrane surface.46 The changes in the chemical shiftsof the protons and carbons of Tyr1, Gly2, Gly3, and Phe4 (Figure4) again support one of our previous papers47 illustrating theSTD (saturation transfer difference) of enkephalins in the GM1micelle. In our study, it is unveiled that GM1 has strong influencein the electrostatic interaction of leucine enkephalin with themembrane surface. The two-dimensional NOESY (not shown)and ROESY (Figures S7-S10 in Supporting Information) spectrawere assessed to see if there is any lipid-peptide NOE, but noNOE or ROE was found between the lipid and the peptide. Thismay be due to the fast tumbling of the peptide leucine enkephalinat the surface of both the bicelles in the NMR time frame indicatingweak interaction of the peptide with both the bicelles.48

Conformation of the Peptide in Water and the Bicelles. Asreported in some recent works on enkephalin in the phos-pholipids membranes,4,13 our structural study demonstrates thatdifferent conformers of leucine enkephalin are possible for thepeptide in the membrane environment (Figure 6). Thirty onepercent of the conformers are random in water, and 69% of theconformers are bent. Therefore, in water the bent structures surpassthe random structures. Three families of structures with twomain conformational patterns, single bent and double bent, arefound for LENK in the PC and GM1 containing PC bicelles,where differences in the groups arise due to the different relativeorientation of tyrosine side chain with respect to the phenylalanine.The resultant bent-like structures can be assigned as turn accordingto the report by Kallick et al.,49 where the requirement for a turnconformation was ascribed as the C1

R-C4R e 8 Å. But, the

turn-like structures here are not the exact �-turn, since the 3JNHCR

coupling constants, 8.0 Hz for Phe4 and 9.0 Hz for Leu5, do notcorrespond the expected magnitudes indicative of the �-turn.Therefore, the structures of LENK in the obtained in the PC andGM1/PC bicelles do not resemble type I, II, III, or IV �-turns

(45) Kimura, T. Biochemistry 2006, 45, 15601–15609.(46) Wishart, S. D.; Sykes, D. B.; Richards, F. M. Biochemistry 1992, 31,

1647–1651.(47) Chatterjee, C.; Majumdar, B.; Mukhopadhyay, C. J. Phys. Chem. B 2004,

108, 7430–7436.(48) Bhattachariya, S.; Domadia, P. N.; Bhunia, A.; Malladi, S.; David, S. A.

Biochemistry 2007, 46, 5864–5874.(49) Watts, C. R.; Tessmer, M R.; Kallick, D. A. Lett. Pept. Sci. 1995, 2,

59–70.

Table 3. Average r(CrTYR-PHE),a re-e,a and r(Cr

GLY-LEU)a of EachGroup for the 90 Lowest Energy Conformations of LENK in

Water, DMPC/CHAPS Bicelles, and DMPC/CHAPS/GM1Bicelles of pH 6.6 (q ) 0.25) at 25 °C

group r(CRTYR-PHE) re-e r(CR

GLY-LEU)

water I 7–8.5 9–11 7–8II 8.5–9 10–12 8–9III 9–10 11–14 7.5–9

DMPC/CHAPS bicelle IV 8–9 10–11 5–7V 6.5–7.5 9–10 8–9VI 8–9 11–12 6–7

DMPC/CHAPS/GM1bicelle

VII 7–8 7–9 4–7VIII 7–9 11–13 7–9IXA 7.5–8 13 8–9IXB 7–9 13 8.5–9.5IXC 7–8 11–12 8–9IXD 7–7.5 10–12 8–9X 8–9 12–13 8–9

a Average distances were determined using Insight II, Silicon Graphics,O2 Workstation.

Table 4. Average Side Chain �1 and �2 Anglesa of Each Groupfor the 90 Lowest Energy Conformations of LENK in Water,DMPC/CHAPS Bicelle, and DMPC/CHAPS/GM1 Bicelle (q )

0.25) at 25 °C

tyrosine phenylalanine

group�1 ((5)

(deg)�2 ((5)

(deg)�1 ((5)

(deg)�2 ((5)

(deg)

water I -64 85, 95 -61 -79, 101II -63 -90, 90 -64 -90, 90III 172 -83, 97 -58 -85, 95

DMPC/CHAPSbicelle

IV -175 -100, 80 -63 -90, 90V 64 -85, 95 -63 -85, 95VI 177 -95, 85 -63 -90, 90

DMPC/CHAPS/GM1 bicelle

VII -64 -90, 90 -64 -85, 95VIII -65 -84, 97 177 -176, 60IXA -65 -83, 96 -63 -67, 113IXB 180 -101, 78 61 -90, 90IXC -64 -90, 90 61 -90, 90IXD -64 -85, 90 62 -90, 90X 60 -75, 10 177 -109, 70

a Average side chain �1 and �2 angles were determined using Insight II,Silicon Graphics, O2 Workstation.


proposed previously for enkephalin in the model membranes.10

Rather, the conformations of LENK in PC bicelles can be definedas bend and the conformers of LENK in the GM1 containing PCbicelles can be defined as turn according to the recent report byChandrasekhar and co-workers,4 where the requirement for bendor turn is r(CR

GLY-LEU) e 1.0 Å (Table 3) with the value ofPhe4 positive for a 5f 2 � turn and negative for a bend at Gly3.Their study identified that, in water, there is continuous transitionfrom turn to bend and vice versa, while in membranes a singleconformation dominates similar to our findings.

Studies that center on the bioactive conformations of theopiate peptides have revealed that at least four main classes(µ, δ, κ, and σ) of opiate receptors are present, which mayalso have distinct subtypes. Although ambiguities in definitionof µ- and δ-active conformations still remain, several recentstudies50,51 on opiate peptides suggest that the tyrosine and thephenylalanine rings are proximal and direct toward the samedirection for the δ-selective conformers, while the tyrosine andphenylalanine rings are away and point toward opposite directionsin the µ-selective conformers. On the basis of this particulargrouping, it can be suggested that, Leu-enkephalin in waterexhibits a 1:1 possibility of the µ- and δ-selective conformers.In the PC and the GM1 containing PC bicelles also, LENK canadopt both µ- and δ-selective conformations, which indicatesthe flexibility and nonspecific binding of the peptide to thereceptors.

The attempts to rationalize the µ- and δ-selective conformersare also done in terms of the tyrosine and phenylalanine �1

angles.52 The comparison of the �1 angles is however complicatedbecause the position of the Tyr1 side chain cannot be reliablypredicted and variations exist.53 Not surprisingly, the positionof tyrosine we found was variable and the orientation ofphenylalanine is highly conserved in gauche– (�1 = -60°). Thetrans (�1 = 180°) and gauche+ (�1 = +60°) dihedrals are alsopossible in the GM1 containing bicelles. The �1 angle ofphenylalanine at the fourth amino acid position supports thepreference of the bent form of the peptide,54 and hence, the sidechain dihedral for only Phe4 is sufficient to evaluate the opiateactivity of the peptide.

In water, most (69%) of the conformers of enkephalin werefound bent as expected according to the previous report.5 Itwas observed that 36% of the conformers are µ-selective and33% of the conformers are δ-selective in water. In zwitterionicphosphocholine bicelles, mainly (81%) the µ-selective con-formers are possible with a small fraction (19%) of theδ-selective conformers. The number of µ-selective conformers(52%) for LENK in the GM1 containing PC bicelles is alsohigher than the number of δ-selective conformations (17%),which suggests that µ-selective conformations are favoredover the δ-type conformations in bicelles even after incor-poration of GM1. As the results indicate, the peptideconformation is more flexible in the GM1/PC bicelles than inthe PC bicelles, but the association is surprisingly strongerin the GM1/PC bicelles than in the PC bicelles (as observedfrom the fluorescence spectroscopy and the deviation ofchemical shifts). Our results strongly exhibit the preferenceof the conformers of leucine enkephalin in boththe bicelles toward µ-selectivity that correlates the observations

reported by Marcotte et al.,13 where methionine enkephalinwas the target peptide. The similar observations obtained inour study for leucine enkephalin offers a common motif ofenkephalins found in the membrane.

Biologically, opiate receptors are mainly found in theperipheral nervous system, in the spinal cord, and in severalregions of the brain where the ratio of µ- and δ-binding siteshas been shown to vary.13 As different cells compose theseparts of the human body, it is expected that their membranecomposition differs. A number of recent NMR studies onantimicrobial peptides have clearly shown that55–58 individualcomponents of cell membrane have a unique role on the structure,dynamics, folding, and topology of biologically active peptides.On the basis of these reports, we believe that our findings regardingthe effect of ganglioside on the leucine enkephalin will be usefulto evaluate the enkephalin-nervous system interaction mech-anism.

Conclusion

In this study, we have investigated the solution structure ofleucine enkephalin in DMPC/CHAPS (1:4) and DMPC/CHAPS/GM1 (1:4:0.3) bicelles. Comparison of the structures of leucineenkephalin in bicelles with its structures in water has exposedthe possibility of both the µ- and δ-selective conformers in eachbicelle, where µ-conformers are favored over the δ-selectiveconformers. Since, the bicelles used by us consisted of truemembrane components phosphocholine, gangliosides (within5–10 mol %), and cholesterol-mimic CHAPS; therefore, it is notfarfetched to believe that, the leucine enkephalin conformationsdiscussed here are similar to the original structures of enkephalinin the natural cell membranes. Our results strongly reinforce thenotion that GM1 incorporated in artificial membranes at thephysiological relevant concentration can regulate the interactionbetween enkephalins and their receptor sites. Moreover, thoughthe effect of CHAPS on the structure of enkephalin is not clearfrom the study, it will however be worth emphasizing that despitepotential changes in the membrane fluidity caused by the use ofCHAPS, the structures identified are similar to the previousfindings,4,13 thus clarifying that there are no strong unexpectedconsequences arising from the membrane rigidity. Thus the resultsprovide new insights into the interaction between the opiatepeptides and membranes and show great potential for investigationof the small peptide-membrane interaction in general. Newapproaches with both the GM1- and cholesterol-doped isotropicand aligned bicelles are invited to shed light on this current fieldin completing our studies.

Acknowledgment. We are thankful to the instrumental facilityof Department of NMR, Bose Institute and Analytical division,Chemgen Pharma Pvt. Ltd. Kolkata for 500 MHz NMR machines.We also thank Mr. Sudipto Kishore Goswami (Chemgen PharmaPvt. Ltd.) for technical assistance in heteronuclear NMRexperiments. The instrumental facility of Saha Institute of NuclearPhysics, Biophysics Division is highly acknowledged for TEMand the instrumental facility of Satyendra Nath Bose NationalCentre for Basic Sciences, Department of Chemical, Biologicaland Macromoleculer Sciences is acknowledged for DLS. A.G.

(50) Groth, M.; Malicka, C.; Czaplewski, C.; Oldziez, S.; Lankiewicz, L.;Wiczk, W.; Liwo, A. J. Biomol. NMR 1999, 15, 315–330.

(51) Mosberg, H. I. Biopolymers 1999, 51, 426–439.(52) Shen, M. Y.; Freed, K. F. Biophys. J. 2002, 82, 1791–1808.(53) Nikiforovich, G. V.; Hruby, V. J.; Prakash, O.; Gehrig, C. A. Biopolymers

1991, 31, 941–955.(54) Mosberg, H. I.; Omnaas, J. R.; Lomize, A.; Heyl, D. L.; Nordan, I.;

Mousigian, C.; Davis, P.; Porreca, F. J. J. Med. Chem. 1994, 37, 4384–4391.

(55) Dhople, V.; Krukemeyer, A.; Ramamoorthy, A. Biochim. Biophys. Acta2006, 1758, 1499–1512.

(56) Dürr, U. H. N.; Sudheendra, U. S.; Ramamoorthy, A. Biochim. Biophys.Acta 2006, 1758, 1408–1425.

(57) Ramamoorthy, A.; Thennarasu, S.; Tan, A.; Lee, D.-K.; Clayberger, C.;Krensky, A. M. Biochim. Biophys. Acta 2006, 1758, 154–163.

(58) Ramamoorthy, A.; Thennarasu, S.; Tan, A.; Lee, D.-K.; Maloy, L. Biophys.J. 2006, 91, 206–216.


is thankful to University Research Fellowship, University ofCalcutta for financial assistance.

Supporting Information Available: Structures of DMPC,CHAPS, and GM1, justification for the choice of q, experimental detailsof dynamic light scattering, 31P NMR and TEM experiments, plots of

DLS, 31P NMR spectra of bicelles, electron micrographs of bicelles,morphological description of GM1 containing bicelles, tables of 13Cchemical shift assignments of bicellar DMPC and CHAPS and NMRspectra (ROESY) of LENK in water and bicelles. This information isavailable free of charge via the Internet at http://pubs.acs.org.

LA704056D


evidence for effect of gm1 on opioid peptide conformation: nmr study on leucine enkephalin in...

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