Proc. Nati. Acad. Sci. USAVol. 89, pp. 4796-4800, June 1992Biochemistry

Metal binding and folding properties of a minimalist Cys2His2 zincfinger peptide

(protein folding/protein structure/metal binding/nucear magnetic resonance)

ScoTT F. MICHAEL, VALDA J. KILFOIL, MICHAEL H. SCHMIDT, BARBARA T. AMANN, AND JEREMY M. BERGDepartment of Chemistry, The Johns Hopkins University, 34th and Charles Streets, Baltimore, MD 21210 and Department of Biophysics and BiophysicalChemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205

Communicated by Richard H. Holm, February 21, 1992 (received for review October 21, 1991)

ABSTRACT A minimalist Cys2His2 zinc finger peptide,Lys-Tyr-Ala-Cys-Ala-Ala-Cys-Ala-Ala-Ala-Phe-Ala-Ala-Lys-Ala-Ala-Leu-Ala-Ala-His-Ala-Ala-Ala-His-Ala-Lys, has beensynthesized. Metal binding studies using Co2+ as a probeindicated that this peptide forms a 1:1 peptide/metal complexwith a dissociation constant comparable to that observed forother zinc finger peptides. At high peptide concentrations, a 2:1peptide/metal complex also forms, with four cysteinates co-ordinated to Co2+. Additional studies with sequence variants inwhich the canonical hydrophobic residues were changed toalanine, or in which one of the residues between the cysteinesand the histidines was deleted, revealed an even more pro-nounced formation of the 2:1 complex over the 1:1 complex. Inaddition, the absorption spectra of the 1:1 peptide/Co2+complexes of the variant peptides are significantly differentfrom those seen for complexes of the parent peptide or those ofmore typical zinc finger peptides. NMR studies revealed thatthe parent peptide folds in the presence of Zn2+ to a structurevery similar to that observed for other zinc finger peptides ofthis class. Taken together, these results suggest that the metal-binding and canonical hydrophobic residues are necessary andsufficient to determine the structure of this class of zinc fingerpeptides.

The amino acid sequence of a protein contains the informa-tion sufficient to determine its three-dimensional structure(1). A variety of experiments have demonstrated that thisinformation is not distributed uniformly but that a smaller setof residues are crucial for correct structure formation. Forexample, the elegant work of Kaiser and Kezdy (2) showedthat simplified (or minimalist) versions of naturally occurringpeptides could be designed and synthesized, retaining manyof the structural (and, in many cases, functional) propertiesof the native materials. This approach has been extended tomore complex oligomeric structures by DeGrado et al. (3).The uneven distribution of folding information has also beendemonstrated by Sauer and coworkers (4) using combinato-rial mutagenesis. Here, a protein sequence is randomized ina number of positions, functional molecules are selected, andtheir amino acid sequences are determined. It has been foundthat some positions are quite tolerant ofa wide range ofaminoacids whereas others have very stringent restrictions. Theseobservations suggest that a large number of sequences thathave only a small subset of residues fixed may fold infunctionally equivalent manners. The Cys2His2 zinc fingersequences typified by transcription factor TFIIIA may rep-resent a naturally occurring set of sequences of this type.After the determination of the sequence of a cDNA clone ofTFIIIA (5), it was noted that the deduced amino acid se-quence contained nine tandem sequences that shared theconsensus (Phe,Tyr)-Xaa-Cys-Xaa2,4-Cys-Xaa3-Phe-Xaa5-

Leu-Xaa2-His-Xaa3,4-His-Xaa2-6 where Xaa represents rela-tively variable amino acids (6, 7). Subsequently, a largenumber of other deduced amino acid sequences have beenfound to contain regions that match this consensus (8-10).A variety of experiments have shown that each of these

sequences folds into a relatively independent structural do-main around a zinc ion bound to the conserved cysteine andhistidine residues (6, 11-14). A three-dimensional structurewas proposed for these domains based on the discovery ofrecurring structural units in crystallographically character-ized metalloproteins that share the positions of the metal-binding and conserved hydrophobic residues with the zincfinger sequences (15). This structure consisted of a (3-strand,a turn, a second (3-strand, a turn, and a helix. NMR studiesof a number of single zinc finger peptides have revealedstructures very similar to one another and to the predictedstructure (13, 14, 16-20). In addition, titration studies usingcertain of these peptides have revealed the formation ofexclusively 1:1 complexes with metal ions such as Zn2+ andCo2+ (12, 20, 21). The dissociation constants for the peptide/Zn2+ complexes are in the 10-9-10-11 M range. To determinewhether the minimal consensus sequence is necessary andsufficient to determine these properties of the zinc fingerdomain, we have designed and synthesized a peptide (calledMZF, for minimalist zinc finger; Fig. 1) in which essentiallyall of the residues represented by Xaa above are alanine.

MATERIALS AND METHODSPeptide Synthesis. Peptides were synthesized, purified, and

reduced as described (20) with use of a Milligen/Biosearch9050 peptide synthesizer. Experiments were performed un-der an atmosphere of 2-5% hydrogen in nitrogen to avoidcysteine oxidation.Metal Binding Studies. Metal ion titrations were performed

as described (20). Spectra acquired in the course of thetitrations were deconvoluted as linear combinations of theinitial and final spectra. The resulting coefficients were fit toa model involving 1:1 and 2:1 peptide/metal complex forma-tion according to Scheme I (see Results). A simplex method(22) that minimized the sum of the squares of the differencesbetween the observed and calculated coefficients was ap-plied. The equilibrium constants K, and K2, the total numberof moles of active peptide present, and four scaling factorswere used as adjustable parameters. The four scaling factorswere necessary to account for the unknown extinction coef-ficients of the two chemical species PM and P2M and thecontributions of both species to the initial and final spectra.Although the concentration of the peptides was estimatedfrom the ultraviolet absorption of the tyrosine, much betterfits could be obtained by allowing a variable peptide concen-tration. The small discrepancies between the measured and

Abbreviations: MZF, minimalist zinc finger; NOE(SY), nuclearOverhauser effect (spectroscopy).

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MZFMZF-CorelessMZF-A8MZF-Coreless-A8

Lys-Tyr-Ala-Cys-Ala-Ala-Cys-Ala-Ala-Ala-Phe-Ala-Ala-Lys-Ala-Ala-Leu-Ala-Ala-His-Ala-Ala-Ala-His-Ala-LysAla Ala Ala

Ala Ala Ala

FIG. 1. MZF peptide and analogues. Metal-binding residues are in bold and the three conserved hydrophobic residues are underlined. Thethree lysine residues were included to enhance stability. In the analogues, only those residues that differ from MZF are shown. -, Deletion.

calculated peptide concentrations can be attributed to pep-tide oxidation and variations of the extinction coefficients oftyrosine in different peptide environments. To estimate er-rors, minimizations were performed with K, or K2/K,2 fixedover a wide range of values. Errors in K, and K2/K12 wereestimated from ranges of these parameters required to in-crease the agreement factor by a factor of 2 (23). Errors in K2were estimated from the K2 values correlated with themaximum and minimum K, values.NMR Studies. NMR samples were prepared with or with-

out 1.5 equivalents of zinc and adjusted to the desired pHwith deuterated Tris (MSD Isotopes). 1H NMR data wereobtained on a Varian XL400 or a Bruker AM600 NMRspectrometer and processed with software supplied with thespectrometers or with FELIX (Hare Research, Woodinville,WA). Two-dimensional NMR experiments were performedby standard methods (24). All two-dimensional NMR exper-iments were performed on a 1.76 mM sample in 90% 'H20/10% 2H20, pH 5.83. The water signal was suppressed bypresaturation. Delays were set to 3 sec due to the longrelaxation times of the MZF-Zn2+ complex.

RESULTSMetal Binding Studies. The MZF peptide contains the

conserved metal-binding and hydrophobic residues charac-teristic of this class of protein domain, with 16 of theremaining 19 residues being alanine. The absorption spec-trum of MZF in the presence of saturating amounts of Co2+is shown in Fig. 2. The position and intensity of the featuresin the ultraviolet as well as the visible region are nearlyidentical to those observed for other Cys2His2 zinc fingerpeptides, including the consensus sequence peptide CP-1(20). An optically monitored titration of MZF with Co02 isshown in Fig. 3. The shape ofthe observed spectrum changedduring the course of the titration. A spectrum with relativelyred-shifted ligand-field bands was observed early in thetitration (i.e., at high peptide/metal ratios). This spectrumsaturated and decayed, giving rise to the spectrum shown inFig. 2 during the course of the titration. In contrast, nochange in spectral shape was observed for other zinc fingerpeptides during titration (12, 20). The red-shifted spectrum isstrikingly similar to that observed for Co2+ complexes with

0.8

0.6

Q4-L 0.40

0.2

0 1-250

four cysteinate ligands (ref. 25; B. A. Krizek, unpublishedwork in our laboratory). These observations were interpretedaccording to Scheme I.

K1PM = P + M

K2P2M =2P + M

Initially, a 2:1 peptide/Co2+ complex forms via coordinationby two pairs of cysteinate ligands. As the ratio of peptide tometal is decreased via the addition of free metal ion, forma-tion of a 1:1 complex is favored. The above model can bereduced to a cubic equation for the free peptide concentra-tion, [P], where Pt is the total peptide concentration and Mtis the total metal concentration:

Kj[P]3 + (K2 + Kj(2Mt- PJ)[P2+ K2(Kl + Mt - P)[P] - PtKlK2 = 0. [1]

From the solutions of this equation the concentration of thefree metal, [Ml, can be derived by using Eq. 2, and theconcentrations of the 1:1 and 2:1 peptide/metal complexescan then be calculated from the equilibrium constant expres-sions.

[M] = MtKlK2/(KlK2 + K2[P] + K,[Pf). [2]

The absorption spectra from the titration experiment could befit using these equations to yield the dissociation constants K,and K2 as well as the absorption spectra of the pure species.As expected, the pure spectra closely resemble the initial andfinal spectra and, hence, the [Co2+Cys2His2] and [Co2+Cys4]species (Fig. 4). The best fit was obtained with K, = 5 x 10-7M and K2 = 4 x 10-11 M2. The close agreement between theconcentrations observed via the deconvolution procedureand those predicted from Scheme I (Fig. 5) confirms theassignment of the initial spectrum to a 2:1 complex, as noother simple model would predict the observed concentrationdependence. Experiments at 7-fold lower peptide concentra-tions display the lower 2:1 complex concentrations predictedby the above model. The goodness of fit was found to besomewhat insensitive to the values of K, and K2 indepen-dently, due to a high degree of correlation between the twoequilibrium constants. The unitless ratio K2/K12 was more

0.25

0.2

u0.15

0.0 0.1

0.05350 450 550 650 750

Wavelength, nm

FIG. 2. Electronic absorption spectrum of an -260 /AM MZFpeptide solution to which a 4.8-fold excess of Co2' has been added.The spectrum of the peptide in a metal-free buffer has been sub-tracted.

o r500 600 700

Wavelength, nm800

FIG. 3. A series of absorption spectra of an -260 ,M MZFpeptide solution with various concentrations of Co2+ (35-368 ,uM).

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Proc. Natl. Acad. Sci. USA 89 (1992)

1000

-1 800

"S0 600E.g 400c.)

0

, 200.IW A I-

500 600 700 800Wavelength, nm

FIG. 4. Derived spectra for the 2:1 and 1:1 complexes of the MZFpeptide with Co2+. These spectra were calculated as linear combi-nations ofthe initial and final titration spectra by using scaling factorsobtained by fitting the titration data to Scheme I.

well determined and was found to be 240. Expressed loga-rithmically, log(K2/K12) = 2.4 + 0.4. For comparison, thespectra of the consensus zinc finger peptide CP-1 are devoidof any features attributable to 2:1 complex formation even athigh peptide concentration and at high peptide/metal ratios.This suggests that K22/K12 > 105 for this peptide. Given thatK, = 5 x 10-8 M for CP-1 (20), this indicates that formationof the 2:1 complex is significantly less favorable for CP-1 (K2> 2.5 x 10-1o M2) than for MZF.To probe the importance of the presence of the conserved

hydrophobic residues and of the conserved length of thecentral loop, three additional peptides were prepared andcharacterized: MZF-Coreless, MZF-A8, and MZF-Core-less-A8 (Fig. 1). Co2+ titrations were performed with each ofthese peptides. The results of fitting these data are summa-rized in Table 1. Behavior qualitatively similar to that seen forMZF was observed in each case. However, several importantquantitative differences were noted. First, the relative extentof 2:1 complex formation was greater for these peptides thanfor MZF. For MZF-A8, which has one ofthe alanine residuesbetween cysteine and phenylalanine deleted, the ratio K2/K12was reduced to 13 [log(K2/K12) =1 1 + 0.2]. For MZF-Coreless, which has the three hydrophobic residues con-verted to alanine but with the normal spacing, this ratio iseven lower, 1.5 [log(K2/K12) = 0.2 ± 0.2]. Removal of analanine from this peptide to produce MZF-Coreless-A8slightly increased the ratio to 2.8 [log(K2/K12) = 0.4 + 0.2].While the derived absorption spectra ofthe 2:1 complexes areessentially identical for the four peptides, the derived spectrafor the monomeric species are significantly different, with the

250

2 200

.3 1 50

)100

u50

0

0 200 400 600Total [Co2+], uM

800 1000

FIG. 5. Plot of the observed concentrations of the 1:1 (e) and 2:1(m) MZF/Co2+ complexes as a function of total Co2+ concentration,overlaid with a plot of the predicted concentrations of the 1:1 (-)and 2:1 (---) complexes for K1 = 5 x 10' M and K2 = 4 X 10-11M2. The 1:1 complex concentrations decrease at high Co2+ concen-trations due to the increased volume of the peptide sample.

Table 1. Logarithms of the equilibrium constants K1 and K2 andthe unitless ratio K2/K12 found from fits of Scheme I to thetitration data

Peptide log K1 log K2 log(K2/K12)MZF -6.4 ± 0.5 -10.4 ± 0.9 2.4 ± 0.4MZF-A8 -6.3 ± 0.4 -11.5 ± 0.6 1.1 ± 0.2MZF-Coreless -5.2 ± 0.2 -10.2 ± 0.5 0.2 ± 0.2MZF-Coreless-A8 -5.6 ± 0.5 -10.8 ± 1.0 0.4 ± 0.2

Values are from the best fits obtained by using a fixed K2/K12varied over a wide range of values. Uncertainties were determinedas described in text.

extinction coefficient of the largest peak in the visible re-duced from the value of 850 M-1cm-1 observed for theparent MZF to <550 M-l cm-1. In addition, the peaks arebroader and slightly blue-shifted. These observations suggestthat the ensemble of structures for these 1:1 peptide/Co2+complexes is more diverse than for that of the parent MZF.Finally, it appears remarkable that the two deletion variantsbind metal ions tightly at all, given that it has been reportedthat removal of the corresponding residues from a peptidecorresponding to one of the two zinc finger domains from theyeast transcription factor ADR1 completely abolished metalbinding (26). However, subsequent experiments in our lab-oratory revealed that the ADR1 deletion peptide does bindmetal ions and shows behavior similar to the deletion variantdescribed herein (Y.-G. Shi, unpublished work).

Structural Studies. NMR structural studies were performedon the Zn2+ complex of MZF. A one-dimensional NMRspectrum of this complex in H20 (Fig. 6) provides strongevidence for the well-structured nature of this species, ascleanly resolved signals are observed for many of the peptideamide groups and significant dispersion is seen for the carbon-bound protons. This spectrum was straightforwardly assignedwith the use of standard methods despite the fact that 16 ofthe26 residues are alanine.The NMR data provide two lines of evidence that this

peptide folds into a conformation very similar to that ob-served for other zinc finger peptides. First, and most directly,the nuclear Overhauser effect spectroscopy (NOESY) crosspeaks observed are those expected for the canonical struc-ture. The NOEs that involve one or more backbone protonsare summarized in Fig. 7. The downfield region of theNOESY spectrum is shown in Fig. 8. Among the featuresobserved is a series of NN(i, i + 1) NOEs involving Ala5-Ala9, corresponding to the turn at the base ofthe 3-sheet, anda similar series from Ala"6 to Ala`- corresponding to the helix.

ppm

FIG. 6. The 600 MHz one-dimensional 1H NMR spectrum of theZn2+ complex of MZF in 90%o H20/10O 2H20, pH 5.83, at 25°C.

* 1:1 complex, observed* 2:1 complex, observed

- 1:1 complex, predicted-- - 2:1 complex, predicted

.A -

.0M -__* . ~.~~a I r- - - -

4798 Biochemistry: Michael et al.

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Proc. Natl. Acad. Sci. USA 89 (1992) 4799

IIAAK L AHAAn KK Y A\ C AE A C A .A .A Es A A K A\ A L A\ A H A+ A\ AR A K

all'o f g 1 LEOM-,1 -

0.5E0. 0 -

z-0V-

x- 1

NNJj _ -1-1.5 - 1 -0.5 0 0.5 1

I.I........Ii

1.. ----1- - -4 .. ...lI.......I.I

I.....

I.....I

.I I......I

i..-.1... i l

FIG. 7. Summary of NOE connectivities observed for MZF. Acrosshatched box or solid line indicates that a NOE was observed,whereas a stippled box or broken line indicates that a cross peak wasobserved but that overlap with other resonances prevented anunambiguous assignment.

Further support for the assignment of a helical structure tothis sequence comes from the observation of many aN(i, i +3) and 13N(i, i + 3) NOEs spanning this same region. Whileoverlap in the NOESY spectrum presented some difficulty,six of these cross peaks spanning the entire helical regioncould be assigned unambiguously. Support for the presenceof the hydrophobic core comes from the presence of NOEsbetween the side chains of Tyr2, Cys4, Phe'1, Leu17, His20,and His24.The second line of evidence for the canonical structure

comes from comparison of chemical shifts of the MZF/Zn2+

9.0 8.5 8.0 7.5Dl (ppm)

7.0 6.5

FIG. 8. Amide region of a two-dimensional NOESY spectrum ofMZF complexed with Zn2+. The spectrum was recorded at 600 MHzin 90%o H20/10O 2H20, pH 5.83, at 250C. Cross peaks correspondingto the NN(i, i + 1) NOE connections are shown for i = 6-9, the turnbetween the p-strands, and for i = 16-25, the helical region.

6.5

MZF (Appm)

FIG. 9. The corrected chemical shifts of Xfin-31 protons versusMZF protons. The chemical shifts were corrected for differences insequence by subtracting the corresponding amino acid chemical shiftvalue from that of a nonterminal residue Xaa in the tetrapeptideGly-Gly-Xaa-Ala (24). Chemical shifts ofthe individual P protons areincluded for the seven conserved residues. The corrected averagesof the , proton shifts are shown for the remaining residues. P-Protondata for Gly5 (and Ala5) are not included.

complex with those obtained for a representative zinc fingerpeptide complex that has had its structure completely deter-mined (28). The differences in chemical shifts from therandom-coil amino acid values (24) for corresponding protonsfor MZF and Xfin-31 are summarized in Fig. 9. A range fromnearly -1.5 to +1.0 ppm difference is seen in each case. Astriking correlation is observed between the corrected chem-ical shifts of the two peptides. This similarity in chemicalshifts indicates that the hydrogen bonding patterns andpositions of the aromatic residues must be very nearly thesame in the two structures. Further examination of thechemical shifts of resonances from MZF/Zn2+ as well as forother zinc finger peptides revealed a correlation between theamide chemical shifts and the secondary structure similar tothat previously noted in other systems (27). This is shown forMZF and Xfin-31 in Fig. 10. The amide resonances forresidues in the helical region (residues 16-26) occur atrelatively high field, whereas those for the 83-strand region(residues 1-10) occur at lower field.

DISCUSSIONWe have synthesized a peptide that has the most conserved

7.0 features of the TFIIIA-like zinc finger domains but hasessentially all of the remaining residues converted to alanine.

7.5 -,=

C'4C8.8.0 0 E

c.0.

9-

zmx

8.5

0.5-

0.

-0.5

- 1

9.0

-1.5- . 1..

-1.5 - 1 -0.5 0

MZF (Appm)0.5 1

FIG. 10. The corrected chemical shifts of the amide protons ofXfin-31 versus the amide protons ofMZF. The solid line correspondsto the best fit with a correlation coefficient of 0.95. The chemicalshifts of the ,8-sheet amide protons (residues 2-10) appear predom-inantly downfield from those ofthe a-helical amide protons (residues16-26).

. .. . . . . I . . . . I . . . . I. . .

0 -NHA - aH-0 - AH 0 A AkbEB - Ring H

AX

00

:A O A fou

aTN(i, i + 3)

f3N(i, i i- 3)

o 0

'7L-'DA--o^°A °£> ~~~~~Oa IA 17L24 H - 25A atO

22A-23Ao -

20H-2A'A19>A -20H

8a-9A 8cBA jS 23A 24H

8A~~~~~8as ~~~21A 22ZA

o as 6 ByC, - 7c

R *~~~~~0o -2-10 0:X -11,13-15 x1K

* -16-26 0

/~~~~~~~~~~~~~~~~~~~~~~~~~~~~~* .

0

.*.

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4800 Biochemistry: Michael et al.

Metal binding studies using Co2' as a probe revealed that thispeptide can bind metal ions with affinities comparable tothose seen for other single zinc finger peptides. The absorp-tion spectrum for the 1:1 complex produced at relatively highmetal ion concentrations is essentially identical to those ofother zinc finger peptide/Co2+ complexes, indicating simi-larity in the metal binding environments. However, thebehavior of MZF differed from that observed for other zincfinger peptides in that a 2:1 complex forms at high peptide tometal ratios. The absorption spectrum of this complex indi-cates that the Co2+ ion is bound tetrahedrally by fourcysteinate residues. A detailed comparison with CP-1, aconsensus zinc finger peptide with a more natural sequence,reveals that this difference in behavior is due to two factors.First, formation of the 1:1 complex for CP-1 is more favor-able, but only by about an order of magnitude. This effect isnot sufficient to explain the absence of detectable 2:1 com-plex formation for the more typical sequence. Thus, a secondfactor-that 2:1 complex formation is at least an order ofmagnitude less favorable for CP-1 than it is for MZF-isnecessary to account for the qualitative difference in behav-ior. This indicates that the natural sequences, which have notbeen shown to have any tendency toward 2:1 complexformation, must have features that destabilize the alternativestructure in addition to having features that stabilize thecanonical monomeric structure. Synthesis and characteriza-tion of several sequence variants that lack the conservedhydrophobic residues or have a single amino acid deletion (orboth) revealed that both of these factors contributed to therelative stability of the monomer over the 2:1 complex.NMR methods have been used to probe the three-

dimensional structure of the monomeric form of MZF/Zn2 .Although some spectral overlap does occur, we were able toassign nearly all of the resonances by using standard two-dimensional methods. The amount of overlap seen in thealiphatic region has prevented us from determining the struc-ture de novo by distance geometry methods. Nonetheless,the NMR results strongly support the hypothesis that MZF/Zn2+ adopts a structure very similar to those observed forother characterized zinc finger peptides. In particular, thepattern of NOEs observed is consistent with the secondarystructure and tertiary contacts expected. Furthermore, noNOEs expected from the structure are unambiguously absentand no unanticipated NOEs are observed. In addition, thecorrected chemical shifts observed closely match those seenin other zinc finger peptide complexes, including Xfin-31/Zn2+. The strong correlation observed includes both back-bone and side-chain protons and represents a completelyindependent line of evidence that supports the structuralsimilarity. Finally, the amide proton shifts themselves forMZF/Zn2+ (as well as for other zinc finger peptide, althoughthis had not been previously noted) are suggestive of thesecondary structural features. Taken together, the NMRresults provide a compelling case that MZF/Zn2+ folds andthat the structure is very similar to those observed for otherzinc finger domains of this class.

CONCLUSIONSA 26-amino acid peptide that has the seven most conservedresidues characteristic of the TFIIIA-type zinc finger do-mains but is otherwise essentially polyalanine has beensynthesized and characterized with regard to its metal bind-ing and structural properties. Remarkably, this peptide foldsin the presence of appropriate metal ions to form a complexthat has a three-dimensional structure very similar to thoseobserved for zinc finger domains with more natural se-quences. However, this peptide differs from natural se-quence peptides in that, under certain conditions, it forms a2:1 peptide/metal complex in which the cysteinate ligands arecoordinated to the metal ion but the histidines are not.

Sequence variants that lack the three conserved hydrophobicresidues or that have one internal residue deleted exhibitincreased 2:1 complex relative to 1:1 complex formation. Inaddition, only the peptide having both the conserved hydro-phobic residues as well as the normal spacing between themetal binding and hydrophobic residues produces a 1:1complex with Co2+ that appears to have a metal ion envi-ronment similar in detail to the natural metal binding sites.These results demonstrate that the information present in thepositions of the seven most conserved residues that charac-terize this family of proteins is necessary and sufficient toproduce the correctly folded structure in an otherwise fea-tureless polyalanine background.

Financial support for this work has been provided by the NationalInstitutes of Health, the National Science Foundation, and theLucille P. Markey Trust. The NMR spectrometer was purchasedwith assistance from the National Institutes of Health. V.J.K. is therecipient ofa scholarship from the Fulbright Foundation, B.T.A. wasthe recipient of a fellowship from the Institute for BiophysicalResearch on Macromolecular Assemblies, and J.M.B. is a Presiden-tial Young Investigator and an Alfred P. Sloan Fellow.

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