doi:10.1006/jmbi.2000.3936 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 300, 1377±1387

The Contribution of Buried Polar Groups to theConformational Stability of the GCN4 Coiled Coil

Hai Zhu1, Scott A. Celinski2, J. Martin Scholtz1,2* and James C. Hu1

1Department of Biochemistryand Biophysics, and2Department of MedicalBiochemistry and Genetics, andCenter for AdvancedBiomolecular Research, TexasA&M University, CollegeStation, TX 77843, USA

Present address: H. Zhu, BioinforPioneer H-Bred, Johnston, IA 50131

E-mail addresses of the corresponjm-scholtz@tamu.edu; jimhu@tamu.

0022-2836/00/051377±11 $35.00/0

The dimeric interface of the leucine zipper coiled coil from GCN4 hasbeen used to probe the contributions of hydrophobic and hydrogenbonding interactions to protein stability. We have determined the ener-getics of placing Ile or Asn residues at four buried positions in a two-stranded coiled coil. As expected, Ile is favored over Asn at these buriedpositions, but not as much as predicted by considering only the hydro-phobic effect. It appears that interstrand hydrogen bonds form betweenthe side-chains of the buried Asn residues and these contribute to theconformational stability of the coiled-coil peptides. However, these contri-butions are highly dependent on the locations of the Asn pairs. The effectof an Ile to Asn mutation is greatest at the N terminus of the peptideand decreases almost twofold as we move the substitution from the N toC-terminal heptads.

# 2000 Academic Press

Keywords: leucine zippers; protein stability; hydrogen bonds; hydrophobicinteraction

*Corresponding author

Introduction

The leucine zipper is a protein dimerizationmotif found in many eukaryotic transcription fac-tors where it serves to bring two DNA-bindingdomains into appropriate juxtaposition for bindingto transcriptional enhancer sequences. Dimeriza-tion of leucine zippers occurs via the formation of ashort parallel coiled coil, with a pair of a-heliceswrapped around each other in a slight left-handedsuperhelical twist (Figure 1). The leucine zipper ischaracterized by a heptad repeat of leucine(Landschulz et al., 1988), where each position inthe heptad is identi®ed following conventionalnomenclature for coiled coils, (abcdefg)n (McLachlan& Stewart, 1975).

Hydrophobic packing is thought to be the majorinteraction between two monomers at their inter-face. The coiled coil is formed by residues at the aand d positions from two monomers packingagainst one another to create a hydrophobic inter-face, conforming to Crick's ``knobs-into-holes''model (Crick, 1953), with each residue at positionsa and d surrounded by four others from the neigh-boring helix. The d positions are almost alwaysoccupied by leucine, and the a positions are usually

matics Department,-0552, USAding authors:edu

comprised of b-branched residues such as Ile, Thrand Val.

One interesting feature of leucine zippers is thepresence of a polar residue (usually Asn) at an aposition within the hydrophobic interface, usuallyin the middle of the leucine zipper domain (Hurst,1994). X-ray crystallographic studies of the leucinezipper from GCN4 (O'Shea et al., 1991) haveshown that this a position Asn residue adopts anasymmetric conformation in the dimer and theside-chains of the two Asn residues from differentmonomers form a single hydrogen bond with eachother. Most naturally occurring leucine zippershave this buried intersubunit hydrogen bondbetween two polar groups in the hydrophobiccore. Substitution of the Asn residue by a Val resi-due in this third a position dramatically stabilizesthe GCN4 coiled coil (Alber, 1992). Thus, it wouldappear that the Asn residue acts as a destabilizingelement for the leucine zipper dimer. It has beensuggested that this destabilization may help tomake the dissociation of the dimer more facile(O'Shea et al., 1991), thereby allowing the redistri-bution of monomers into different arrays of homo-dimers and heterodimers needed to modulate therange of genes affected by this class of transcrip-tion factors (McKnight, 1991). Although this buriedhydrogen bonding interaction is likely to be ener-getically less favorable than the hydrophobic inter-actions from valine or isoleucine side-chains, itplays an important role in determining the dimeri-

# 2000 Academic Press

Figure 1. A depiction of the leucine zipper of GCN4arranged as a parallel coiled coil. (a) Side view of thedimer. The amino acid backbones in a helical confor-mation are presented by cylinders with the path of thepolypeptide chain indicated by the broken line. Side-chains are represented as knobs, with the targeted a pos-itions highlighted. (b) End-view showing how differentheptad positions are arranged in the dimer. The side-chains at the a positions are buried in the dimer inter-face. (c) Sequence of the leucine zipper GCN4 wild-typeand mutants constructed. The sequence shown is that ofthe wild-type peptide. Lowercase letters over thesequence indicate positions in the heptad. The a pos-itions altered here are highlighted. The numberingscheme employed is intended to maintain consistencywith previous GCN4 peptides (O'Shea et al., 1989). Allthe mutants are listed below the wild-type sequence,with the corresponding mutated a positions shown. Thenomenclature of the mutants are based on the sequenceof the mutated a positions.

1378 Buried Polar Groups in Leucine Zippers

zation speci®city and makes the leucine zipperform a unique parallel homodimer. Changing theburied Asn residue to either a Val residue or a Leuresidue leads to the formation of mixtures ofdimers and trimers (Harbury et al., 1993; Potekhinet al., 1994).

Since both hydrophobic packing and hydrogenbonding are involved in the dimerization inter-

action, leucine zippers are a good model to deter-mine the effects of hydrophobicity and hydrogenbonding on stability. To accomplish this goal, wecompared the thermodynamic stabilities of mutantGCN4 leucine zippers with different combinationsof Asn or Ile residues at four different a positions.By measuring the differences in stability betweenthese mutants using thermal denaturation, weattempt to determine the contribution of hydro-phobicity and hydrogen bonding to the dimeriza-tion stability of the leucine zipper coiled coil.

Results

Design of a position mutants of the GCN4leucine zipper

To investigate the effects of the hydrophobicityand the hydrogen bonding capability of a buriedresidue on the dimerization stability of the GCN4leucine zipper, a series of a position mutants wasdesigned and analyzed. Figure 1 presents a cartoonof the wild-type GCN4 leucine zipper dimer withthe a positions highlighted. There are ®ve a pos-itions in the GCN4 leucine zipper: positions 2, 9,16, 23 and 30. Together they form the core of thedimeric interface, along with the four residues atthe d positions. The amino acid sequence for the®ve a positions in the wild-type GCN4 leucine zip-per is Met, Val, Asn, Val, Val, while the d positionsare all occupied by Leu residue.

Here, we report an analysis of the last four apositions in GCN4 to provide a comparison to ourprevious study at these positions (Zeng et al.,1997). The amino acid substitutions in the mutantsused are shown in Figure 1. The approach we haveemployed is to place various numbers of Asn resi-dues in the peptide at different a positions, and ®llthe other a positions with Ile residues. The mutantscan be divided into two sets: one set includes themutants with only one Asn residue but at differenta positions; the other set contains those mutantswith various numbers of Asn residues. Themutants in the ®rst set were designed to character-ize the position effect of the interhelical Asn hydro-gen bonding on the stability of the leucine zipperdimer, while the second set was used to explorethe effect of increasing the total number of Asnpairs on the overall conformational stability of thecoiled-coil dimer.

Structural characterization of the aposition mutants

The peptides were constructed, expressed andpuri®ed as described in Experimental Procedures.Of the 16 possible variants, ®ve did not expressand could not be studied; here we report the prop-erties of the remaining 11 peptides. If these pep-tides form the expected two-stranded coiled-coilstructure, they should have the following charac-teristics: (i) the CD spectra of the peptides shouldhave double negative minima at 207 nm and

Buried Polar Groups in Leucine Zippers 1379

222 nm, indicating the formation of a-helical struc-ture; (ii) the apparent molecular mass of the pep-tides in aqueous buffer measured by sedimentationequilibrium should be that of a dimer; (iii) in dena-turation studies, the apparent stability of the pep-tides should be concentration dependent, asrequired for a monomer-dimer equilibrium.

The CD spectra of all the a position mutant pep-tides were taken from 200 nm to 320 nm at pH 7.0,0 �C at a total peptide concentration of 10 mM (datanot shown). All the peptides except NNNN exhib-ited typical a-helical CD spectra. The CD spectrumof NNNN, on the other hand, exhibited a mixtureof random coil and a-helical structure. The ellipti-city at 222 nm (Table 1) obtained for most of thepeptides was ÿ32,000(�1500) deg cm2 dmolÿ1, cor-responding to about 95 % a-helical structure. How-ever, under the same conditions, NINI, NNNI andNNNN showed only 78 %, 73 % and 46 % helix,respectively, which suggests they form less stablea-helices. After increasing the total concentration to1 mM, the a-helicity of NINI and NNNI reach100 %, but the helicity of NNNN is still only �75 %(data not shown).

The oligomerization states of the peptides weredetermined by sedimentation equilibrium at 4 �C ata peptide concentration of 40 mM. The data fromall the mutants can be best described by either asingle dimer species model or a monomer-dimerequilibrium model (Table 1). A typical example isshown in Figure 2 for the IINI variant, which canbe described by the single dimer species model.Based on these results, all of the a position mutantsof the GCN4 leucine zipper form dimeric coiled-coil structures at 4 �C, although their level of stab-ilities are very different.

Thermodynamic stability of the aposition mutants

The stability of each of the peptides was deter-mined by measuring the ellipticity at 222 nm as afunction of temperature from 0 to 100 �C. The ther-

Table 1. Structural characterization of the a position variants

Variant[y]222

(deg cm2 dmolÿ1)

VNVV ÿ31,400IIII ÿ32,220NIII ÿ31,490INII ÿ31,100IINI ÿ31,670IIIN ÿ29,100NINI ÿ29,700INNI ÿ30,000ININ ÿ30,410IINN ÿ31,700NNNI ÿ26,430NNNN ÿ18,410

The peptides are named as shown in Figure 1C. The [y]222 valuespH 7.0.

a The apparent molecular mass was determined from the analysis

mal denaturation of every mutant was performedat ®ve concentrations: 2.5, 5, 10, 20 and 40 mM(monomer). The reversible thermal unfolding formost of the peptides follows a simple cooperativeunfolding event that can be described by a two-state model identical to that observed for the wild-type GCN4 leucine zipper (Thompson et al., 1993).The peptides that follow this simple behaviorinclude INII, IINI, NINI, ININ, IINN, INNI andNNNI (Figure 3(a)) (the peptide NNNN alsoappears to fall into this class, but its lack of stab-ility precludes a de®nitive description). The ther-mal unfolding of IINI as a function of peptideconcentration is shown in Figure 3(b) as a repre-sentative case. As illustrated in Figure 3(b), thetransition midpoint temperature, Tm, increases asthe peptide concentration increases, consistent witha model in which the unfolding transition is tightlycoupled to the dissociation of the dimer. However,the unfolding curves for one of the peptides, NIII,do not appear to adhere to two-state behavior, butrather this peptide appears to unfold in at leasttwo stages (Figure 3(c)). For this reason, this pep-tide will not be considered further in this studyand a characterization of its thermal unfolding willbe described elsewhere (H.Z. et al., unpublishedresults). The levels of stability of IIII and IIIN arethe greatest of all the mutants: at a peptide concen-tration of 20 mM, both are partially folded, even at100 �C (Figure 3(a) and (c)). In contrast, NNNN isthe most unstable peptide; the peptide is not com-pletely folded at 0 �C even at a peptide concen-tration of 1 mM (data not shown). Therefore, wecould not assess the thermodynamic stability ofeither IIII, IIIN or NNNN and they were omittedfrom further analysis.

A cooperative two-state model that couplesunfolding and dissociation (equations (6)-(10)) was®t to the observed thermal unfolding data for theGCN4 variants, using all ®ve peptide concen-trations. In the analysis of our data, several par-ameters were found not to vary between thedifferent peptide variants. These parameters, �CP

of GCN4

Calculated MM(monomer, Da)

Apparent MM(Da)a

4161 81004202 88004203 85004203 83004203 88004203 87004204 80004204 82004204 82004204 84004205 78004206 5100

are reported for a total peptide concentration of 40 mM, 0 �C and

of analytical ultracentrifugation studies as described in the text.

Figure 2. Sedimentation analysis of the a positionmutant IINI. The total peptide concentration was 40 mMin terms of monomers in 10 mM potassium phosphatebuffer, 200 mM sodium chloride (pH 7.0), at 4 �C. Theabsorbance was monitored at 225 nm. The curve shownrepresents the dimer single species model.

1380 Buried Polar Groups in Leucine Zippers

and a, were therefore treated as common par-ameters and iteratively determined for the entiredata set. The remaining parameters unique to eachpeptide are shown in Table 2 and the calculatedcurves for IINI based on these parameters areshown in Figure 3(b). It can be seen from Table 2that the stability from most stable to least stableof the mutants is IINI > INII > IINN > INNI >ININ > NINI > NNNI. As more Asn residues areadded at the a positions, the conformationalstability decreases, consistent with the idea that thehydrophobicity of the residue at the buried a pos-ition is important. In addition to the number ofAsn residues present, there is a pronounced pos-ition-dependency to Asn substitution on the stab-ility of the peptides. Mutants IINI and INII bothcontain a single Asn residue, but it is located atdifferent a positions. IINI is more stable than INIIby 1.8 kcal molÿ1. Similarly, for those mutants con-taining two Asn residues at different a positions,IINN is more stable than ININ, INNI and NINI by1.8, 2.0, 2.2 kcal molÿ1, respectively.

The destabilization of Ile to Asn replacementsis position dependent

There is a redundancy in our data set that allowsus to determine the change in conformational freeenergy for an Ile to Asn substitution at each of thea positions in several different backgrounds. Table 3illustrates this point. For example, at position 9 inthe peptide we have two sets of peptides that canbe used to assess the differences between Ile and

Asn: either IINI and NINI or INNI and NNNI.Likewise, we have a pair of peptides that can beused for the substitution at a position 30, three setsof peptides at a position 16 and a single pair ofpeptides for a position 23. The results from thedifferent comparisons at an individual a positionare all in (Table 3).

Surprisingly, we ®nd almost a twofold differencein the change in the Gibbs free energy of unfolding(��G) for an Ile to Asn replacement at different apositions in the coiled coil. The effect of an Ile toAsn mutation on the free energy of unfolding islargest at the N terminus of the peptide anddecreases almost twofold as we move the substi-tution from the N to C-terminal heptads.

Discussion

One important issue in protein folding is thedelineation of the energetic contributions of hydro-gen bonding and hydrophobic packing to globularprotein stability. The importance of hydrogenbonding to protein stability, chie¯y through is rolein stabilizing secondary structure, was ®rst empha-sized by Pauling (Pauling & Corey, 1951; Paulinget al., 1951). Kauzmann (1959), and later Tanford(1962, 1979), pointed out the importance of thehydrophobic effect to the stability of proteins, andthis effect gained prominence as the key force pro-viding stability to globular proteins. With theadvent of mutational studies over the last tenyears, it has become clear that hydrogen bondingand the hydrophobic effect are clearly the twodominant non-covalent forces stabilizing proteins,and Pace has suggested that they make almostequal contributions (Myers & Pace, 1996; Pace,1992; Pace et al., 1992; Shirley et al., 1992). Onedrawback of using mutational studies in proteinsto address these issues is that, in general,mutations will leave cavities in proteins and poss-ibly unpaired polar groups. Even with detailedstructural data, it is often dif®cult to account com-pletely for these effects. The homodimeric coiled-coil structure provided by the GCN4 leucine zipperpeptide is in many ways a better suited and sim-pler system for performing mutagenesis exper-iments. For example, when we perform an Ile toAsn mutation, the change is made in both peptidesand no single polar group is left buried. Modelbuilding suggests that good packing is stillachieved and no voids are left. Thus, these simplepeptides provide an excellent vehicle for investi-gating the relative contributions of hydrogen bond-ing and the hydrophobic effect to proteinconformational stability.

Because the folding of dimeric GCN4 leucinezippers is coupled with their dimerization, thefolded and unfolded fractions of the proteindepend on the total peptide concentration used ina denaturation experiment. Thus, the unfolding ofthe GCN4 leucine zipper dimer can be controllednot only by temperature, but also by the peptide

Figure 3. Thermal denaturationexperiments of the a positionmutants monitored by circulardichroism at 222 nm in 10 mMphosphate buffer. (a) The thermaldenaturation curves of the a pos-ition mutants ®tted to a 2-stateunfolding model: NNNN (*);NNNI (&); NINI (}); ININ (~);INNI (�); IINN (�); INII (*); IINI(&) and IIII (~). The peptide con-centration was 20 mM total in termsof monomers except the NNNNwhich was 40 mM. (b) The thermaldenaturation curves of IINI at pep-tide concentrations of: 2.5 mM (*);5.0 mM (&); 10 mM (}); 20 mM (�)and 40 mM (~). The curves on theplot are generated from the par-ameters listed in Table 2. (c) Thethermal denaturation curves of thea position mutants with three-stateunfolding at 40 mM: IIIN (*); NIII(&).

Buried Polar Groups in Leucine Zippers 1381

concentration. By varying protein concentrations,multiple data sets can be obtained and simul-taneously analyzed. The advantage of thisapproach is that multiple data sets can give usboth a well-de®ned pre-transition and post-tran-sition baseline, even though some of the individualcurves may not show good baselines. Furthermore,thermodynamic parameters can be determined to a

higher level of precision than from any individualcurve by performing a global ®t to multiple datasets.

Here, a series of thermal unfolding curves ofpeptides was obtained at identical solution con-ditions, using changes in peptide concentration toalter the Tm (and �Hm). The ®t of the dimericmodel derived from equation (1) to the data (see

Table 2. Thermodynamic parameters for the thermal unfolding of the a position variants of GCN4

VariantTm (�C)40 mM

T0

(�C)�H0

(kcal molÿ1)�G (60 �C)

(kcal molÿ1)

VNVV(wt) 56.1 � 0.1 98.4 � 0.1 66.3 � 0.1 6.2 � 0.2IINI 79.5 � 0.1 122.1 � 0.8 73.2 � 1.6 9.9 � 0.2INII 69.5 � 0.1 113.8 � 1.1 66.8 � 0.8 8.1 � 0.1IINN 64.0 � 0.1 104.9 � 0.8 69.9 � 0.4 7.3 � 0.1INNI 52.5 � 0.1 95.6 � 0.4 62.6 � 0.8 5.5 � 0.1ININ 50.6 � 0.1 93.8 � 1.5 62.3 � 0.3 5.3 � 0.1NINI 45.0 � 0.7 99.1 � 1.4 55.2 � 1.8 5.1 � 0.1NNNI 21.2 � 0.5 63.8 � 0.8 54.1 � 1.0 0.6 � 0.1

For each of the a position variants of GCN4, the midpoint of the thermal transition (Tm) obtained at a total peptide concentrationof 40 mM is given as well as T0 and �H0, the temperature and enthalpy change where �G � 0. These latter parameters, which areindependent of peptide concentration, are determined as described in the text. From T0 and �H0, a values of �G can be determinedat 60 �C with the standard condition of 1 M total peptide concentration. For the determination of �G(60 �C), a constant value for�CP of 0.31 kcal molÿ1 Kÿ1 was employed (see the text). The error on Tm was determined from the individual unfolding curves andthe errors on the other parameters are the standard deviations obtained from the global analysis as described in the text.

1382 Buried Polar Groups in Leucine Zippers

Figure 3(b), for example) supports the use of themodel and allows us to obtain the thermodynamicparameters shown in Table 2 for the variants. Theresults of the analytical ultracentrifugation studies(Figure 2 and Table 1) are also consistent with thedimeric nature of the unfolding transition, as illus-trated in equation (1).

Since �CP is proportional to the hydrophobicsurface area buried during the folding of proteins(Murphy et al., 1992; Myers & Pace, 1996; Spolar &Record, 1994), it is expected that �CP shouldbecome progressively larger as the number of Asnresidues decreases. However, the �CP valuesobtained from our denaturation experiments werevery small even for the most hydrophobic mutant,and any differences in �CP among the mutant pep-tides were too small to detect. Therefore, we usedan average value of �CP for all the mutants. Sincean extrapolation is required, the values of T0 and�H0 are slightly dependent on the value used for�CP. In contrast, the values of �G(60), the freeenergy of unfolding at a temperature in or near the

Table 3. The heptad dependence to the conformational stabil

a Position��Gobs (60 �C)

(kcal molÿ1)a

9 4.8 � 0.24.9 � 0.2

16 4.5 � 0.24.4 � 0.2

23 2.6 � 0.230 2.6 � 0.2

2.8 � 0.2

a ��Gobs is the difference in conformational stability from the datsents the change in stability for an Ile to Asn replacement. Positive v

b ��Gobs ÿ ��GHé corrects for the difference in the level of hydrthe text). The negative values indicate that the Asn-containing peptibic effect, and therefore represents the stabilizing contribution of burface.

transition region for these peptides, is not asdependent on �Cp. Therefore, the most meaningfulway to compare the conformational stabilities ofthe peptides is by comparing �G at 60 �C.

Peptide INII has a single Asn residue at a pos-ition 16, identical to that found in the wild-typeGCN4 (VNVV in our nomenclature), however INIIdiffers from VNVV by having Ile residues insteadof Val residues at the other a positions. The ther-modynamic stability of VNVV was measuredusing the same approach as for the other mutants(Table 2). T0 and �H0 obtained for VNVV are98.4 �C and 66.3 kcal molÿ1 per dimer, which arevery similar to previous results obtained on arelated variant of the GCN4 leucine zipper(Thompson et al., 1993). The INII variant is morestable than VNVV (��G � 1.9(�0.2) kcal molÿ1 at60 �C), consistent with previous results in a differ-ent model dimeric coiled coil that show that longerb-branched side-chain residues at the a positionsare more stable (Zhu et al., 1993). Most of thisdifference in stability can be reconciled by consid-

ity of Ile to Asn mutations

��Gobs ÿ ��GHé

(kcal molÿ1)b Variants used

ÿ0.8 � 0.2 IINI! NINI

ÿ0.7 � 0.2 INNI! NNNI

ÿ1.1 � 0.2 IINI! INNI

ÿ1.2 � 0.2 NINI! NNNI

ÿ3.0 � 0.2 INII! INNI

ÿ3.0 � 0.2 IINI! IINN

ÿ2.8 � 0.2 INII! ININ

a in Table 2 for the indicated pairs of peptides. This value repre-alues indicate that the Ile containing peptide is more stable.ophobicity between Ile and Asn at a site that is 85 % buried (seede is more stable than expected, based on the simple hydropho-ied hydrogen bonding between Asn residues in the dimer inter-

Buried Polar Groups in Leucine Zippers 1383

ering the differences in hydrophobicity betweenVal and Ile at this mostly buried site using theoctanol to water transfer free energies (�Gtr)described by FaucheÂre & Pliska (1983).

In contrast to the differences between Val and Ileat the a positions, the effects of replacing a buriedIle with an Asn are very dramatic. The peptidewith all four a positions occupied by Ile (IIII) is sostable that we cannot observe a thermal unfoldingtransition below 100 �C, even at low peptide con-centrations. On the other hand, the NNNN peptideis not completely folded at 0 �C at a concentrationof 1 mM. Table 2 shows that as we replace buriedIle residues with Asn residues, the level of stabilitydrops. Furthermore, there is a strict additivity tothe energetics of the Ile to Asn substitutions at thea positions in our peptides. As an illustration,Figure 4 shows a mutant cycle relating the pep-tides IINI and NNNI through the two single var-iants INNI and NINI. This result suggests thatthere is no energetic coupling between the a pos-itions and that they behave as independent sites.This same result is often observed in globular pro-teins (Wells, 1990), suggesting that there is nothinginherently unique in the equilibrium behavior ofthe coiled-coil structure that would preclude itsuse as a model for protein folding and stability.

There is a striking difference in energetics for anIle to Asn replacement at positions 9 and 16 com-pared with the same replacement in the C-terminalheptads (positions 23 and 30) (Table 3). For pos-itions 9 and 16, we ®nd 4.8 and 4.4 kcal molÿ1

differences in stability after the substitution froman Ile to an Asn residue. In contrast, the C-terminalheptads have a smaller energy difference, 2.6 and2.8 kcal molÿ1, after the Ile to Asn replacment.

Two recent reports also suggest that the C-term-inal heptads have different properties than theother heptads. It has been suggested that the

Figure 4. A double mutant analysis of the energydifference between the Ile residue and the Asn residueat the a positions 9 and 16. The ��G values for theindividual a position variants are calculated as shown inTable 3.

region of GCN4 encompassing residues 17 through29 acts as a ``trigger sequence'' that mediatesdimerization (Kammerer et al., 1998). The triggersequence pattern has been found in many othercoiled coils and appears to be required for theassembly of GCN4 and other coiled coils. Inaddition, Myers & Oas (1999) present an argumentthat the C terminus of the GCN4 leucine zipper iscapable of transient monomeric helix formationand that this intermediate is important in the kin-etic mechanism for the folding of GCN4.

One can estimate the contributions of the hydro-phobic effect to ��G for the Ile to Asn substi-tutions, using the model compound transfer freeenergies described by FaucheÂre & Plishka (1983)and a calculated burial of 85 % for the side-chainsat the a positions (based on model building fromthe crystal structure of the GCN4 leucine zipper).With these assumptions, the hydrophobic effectwould contribute about 5.6 kcal molÿ1 of thedifference in stability between an Ile pair and anAsn pair. The use of alternative hydrophobicityscales, such as those derived from water to cyclo-hexane transfer studies (Radzicka & Wolfenden,1988), would make the differences between Asnand Ile even larger. This estimation of the differ-ence in hydrophobicity is larger than the observedvalues of ��G by 0.7, 1.1, 3.0 and 2.8 kcal molÿ1

for substitutions at positions 9, 16, 23 and 30,respectively. This deviation between calculatedand observed values suggests favorable inter-actions in the dimer involving the Asn residuesthat mitigate the unfavorable effects of a non-polarto polar substitution in a largely buried environ-ment. Differences in helix propensity are also unfa-vorable for an Ile to Asn substitution (Pace &Scholtz, 1998), although this difference is consider-ably smaller in magnitude. Using the helix propen-sity scales derived from a model coil coil (O'Neil &DeGrado, 1990), a single Ile to Asn substitutionwould be expected to be only 0.16 kcal molÿ1 lessstable.

We envision at least two possible sources for thefavorable contribution of buried Asn residues tothe stability of the coiled coil. First, hydrogenbonding between Asn side-chains could be energe-tically favorable. Second, although the side-chainsare mostly buried, the polar functional groups ofthe Asn residues could make favorable interactionsinvolving water and/or other donors or acceptorsin the peptide. Indeed, in the crystal structure ofthe GCN4 leucine zipper one of the Asn16 side-chains is involved in a water-mediated hydrogenbond with Lys15 (O'Shea et al., 1991). The Lys atposition 8 could make a similar interaction with anAsn pair at position 9.

Subtracting the expected contribution of thehydrophobic effect magni®es the context depen-dence of the energetic effect of replacing an Ile pairwith an Asn pair. It should also be noted that theinteractions between ai and giÿ1 positions do notseem to account for the context dependence of��G for the Ile to Asn substitution. The deviations

1384 Buried Polar Groups in Leucine Zippers

from the expectations of transfer free energies aregreater for the a positions in the two C-terminalheptads, where the giÿ1 positions are occupied byGlu22 and Leu29, respectively. If Asn residues atthese positions can make interactions, they wouldhave to be more favorable than the Asn-H2O-Lyshydrogen-bonding network seen around Asn16 inthe crystal structure.

In the wild-type GCN4 coiled coil (VNVV), theAsn residues at a position 16 make a single inter-strand hydrogen bond. This side-chain hydrogenbond is thought to impose speci®city to alignmentof the coiled-coil dimerization by forcing the polara position Asn residues to line up and thus keepthe ``register'' of the structure intact (O'Shea et al.,1991, 1989). Energetically, this single buriedside-chain to side-chain hydrogen bond mustcontribute to the stability of the dimeric complex,and our results suggest that the hydrogen bond isstabilizing.

The role of buried hydrogen bonding to stabilityof globular proteins remains a controversial sub-ject. Two recent theoretical studies suggest thathydrogen-bonded polar groups do not make afavorable contribution to protein stability (Honig &Yang, 1995; Lazaridis et al., 1995), yet recent exper-imental studies, using the mutational approach,reach the opposite conclusion (for a recent review,see Myers & Pace (1996)). As stated above, thedimeric coiled-coil motif has certain advantagesover globular proteins, namely the ability toremove or introduce a pair of polar groups in asingle mutational event, thus obviating the poten-tial problems of leaving unpaired polar groupsburied in the interior of a protein. Since theobserved differences between the stability of a pairof peptides that differ by a single Ile to Asn substi-tution cannot be accounted for by the hydrophobiceffect, it appears that hydrogen bonding plays arole in stabilizing the coiled-coil structure in theGCN4 leucine zipper.

In conclusion, we have determined the ener-getics of placing Ile and Asn residues at a buriedposition in a two-stranded coiled coil. As expected,Ile is favored over Asn at this buried position, butnot as much as expected by considering only thehydrophobic effect. It appears that interstrandhydrogen bonds form between the side-chains ofthe buried Asn residues and these contribute to theconformational stability of the coiled-coil peptides.Thus, our data are consistent with the structuraldata on the GCN4 leucine zipper peptide thatreveal a single interstrand hydrogen bond betweenAsn residues at position 16 in the sequence. On theother hand, our data suggest that the energeticcontributions of hydrogen bonds formed by Asnresidues in the a positions of the two N-terminalheptads are very different from those of the twoC-terminal heptads. In all cases, the hydrogenbonding pattern between the Asn residuesprovides not only speci®city to the alignmentof the coiled-coil dimer, but also adds to theconformational stability.

Experimental Procedures

Peptide expression and purification

All the recombinant peptides were expressed inEscherichia coli strain BL21(DE3) using a modi®ed T7expression system. SalI/BamHI fragments encoding thewild-type and mutant GCN4 leucine zippers weresubcloned from plasmids as described (Zeng et al.,1997) into the same sites in pHZ3 (GenBank accession:AF168612). pHZ3 is a modi®ed version of the T7expression vector pET3a (Studier et al., 1990). Nucleo-tides around the translational initiation site werealtered to give the expressed N-terminal sequenceMST which contains a SalI site in the DNA. The N-terminal methionine is removed by E. coli from pep-tides expressed from this vector. Sequences of theindividual mutants were all determined and con®rmedby DNA sequencing.

Cultures were grown overnight from a single colonyin 10 ml LB medium containing 200 mg/ml ampicillin,then transferred to 1 l LB medium containing 200 mg/mlampicillin and 2 g/ml glucose. When the absorbancy ofthe culture reached 0.5 at 600 nm, 100 mg/l of isopropyl-b-D-thiogalactopyranoside was added to induce theexpression of the T7 RNA polymerase, which transcribesthe gene encoding the leucine zipper peptide. After threehours, cells were harvested by centrifugation and lysedby the use of a french press in 50 mM Tris-HCl and1 mM EDTA (pH 8.0). The pH of the lysate was reducedby adding formic acid to a ®nal concentration of 20 %(v/v). The resulting mixture was centrifuged to removeprecipitated material. The supernatant, which containedour desired peptide, was dialyzed overnight against10 mM sodium acetate, 50 mM NaCl and 1 mM EDTA(pH 4.0), then loaded onto a Source-S column (Pharma-cia). The peptide was eluted off the column by a NaClgradient from 50 mM to 1 M in the same buffer. Thefractions containing the peptide were pooled, and loadedonto a Resource-RPC reverse phase column (Pharmacia).The peptide was eluted using a gradient developed from95 % (v/v) water, 5 % (v/v) acetonitrile, 0.1 % (v/v)tri¯uoroacetic acid to 5 % (v/v) water, 95 % (v/v) aceto-nitrile, 0.1 % (v/v) tri¯uoroacetic acid. The fractions con-taining the peptide were pooled and lyophilized. Thepeptide obtained in this fashion was more than 98 %pure, based on SDS-PAGE and Coomassic blue staining,and its molecular mass was con®rmed by MALDI-TOFmass spectroscopy. The peptide was stored at ÿ20 �C aspowder.

Circular dichroism measurements

CD spectra were acquired on an Aviv 62DS spectropo-larimeter equipped with a thermoelectric temperaturecontrol and stirring unit. Samples were prepared in10 mM potassium phosphate buffer (pH 7.0) and thepeptide concentrations were determined by tyrosineabsorbance in 6 M guanidine hydrochloride using anextinction coef®cient of 1455 Mÿ1 cmÿ1 at 275 nm (Paceet al., 1995). Thermal denaturation was measured bymonitoring the ellipticity change at 222 nm as the tem-perature was increased from 0 to 100 �C in steps of 1 �Cwith an equilibration time of 1.0 minute and a data col-lection time of 30 seconds. All the reported temperaturesare for the cuvette jacket as calibrated by the manufac-turer of the unit. Cuvettes with path length of 10 mmwere used for samples at low concentration (< 50 mM),otherwise cuvettes with path lengths of 1 or 5 mm were

Buried Polar Groups in Leucine Zippers 1385

used. The reversibility of the thermal transition wasdetermined by monitoring the return of the CD signal at222 nm upon cooling from 100 to 0 �C immediately afterthe conclusion of the thermal denaturation experiment.In all cases the transitions were > 95 % reversible.

Determination of the oligomerization states bysedimentation equilibrium

Sedimentation equilibrium measurements were per-formed on a Beckman XL-A ultracentrifuge at 4 �C.Samples were dialyzed against 10 mM potassium phos-phate, 200 mM sodium chloride (pH 7.0), and analyzedat a concentration of 40 mM with a rotor speed of46,450 g. The absorbance at 225 nm was monitored andequilibrium was reached after 36 hours. Partial molarspeci®c volumes and solvent densities were obtained asdescribed by Laue et al. (1992). The data were analyzedwith software provided by Beckman.

Data analysis

For a dimeric two-state unfolding transition, the equi-librium between native dimer (N) and unfolded mono-mers (2U) is:

N2 $Kd2U �1�

where Kd � [U]2/[N2]. Given that the total peptide con-centration, Pt, in terms of monomers is Pt � 2[N] � [U],the concentration of unfolded monomer, [U] can beexpressed as:

�U� �������������������������K2

d � 8KdPt

qÿ Kd

4�2�

Thus, the fraction of each species is de®ned as:

fN � 2�N2�=Pt �3�

fU � �U�=Pt �4�

fN � fU � 1 �5�where fN is the mole fraction of molecules in the foldedstate, and fU is the mole fraction of the molecules in theunfolded state. By combining equations 2 and 4, one®nds:

fU �������������������������K2

d � 8KdPt

qÿ Kd

4Pt�6�

The observed CD signal, Y0, can be described in termsof native and unfolded baselines, YN and YU, respect-ively, and the fraction of unfolded molecules by theexpression:

Y0 � �YU ÿ YN�fU � YN �7�where YU was found to be constant at temperatureshigher than the melting region for all the peptidesstudied. YN was approximated by a linear function oftemperature:

YN � YN�0� � aT �8�

where YN(0) is the signal of baseline at 0 �C, and a is theslope of the pre-transition baseline. The equilibrium con-stant, Kd can be expressed in terms of thermodynamicparameters as:

Kd � exp�ÿ�G0=RT� �9�

Kd � exp�H0

R

1

T0ÿ 1

T

� ��

��Cp�T0 ÿ T � T � ln�T=T0��RT

��10�

where the reference temperature T0 is the temperature atwhich the intrinsic free energy difference �G is equal tozero and independent of the peptide concentration(Thompson et al., 1993). Likewise, �H0 is the enthalpychange at temperature T0 and is also concentration inde-pendent. For our analysis, we have assumed that �Cp isindependent of temperature over the temperature rangeexamined following standard practice (Becktel &Schellman, 1987; Makhatadze & Privalov, 1995;Thompson et al., 1993).

The thermodynamic parameters were determined froma global analysis of all the thermal denaturation curves atdifferent peptide concentrations with equations 6, 7, 8and 10 using non-linear least-squares ®tting routines inIgor Pro (WaveMatrics). In the initial ®tting process, all ofthe thermodynamic parameters, as well as a and YN(0),were allowed to vary. The slopes of the pre-transitionbaseline (a) resulting from this process are identicalwithin error, so a ®xed slope a � 78.7 deg cm2 dmolÿ1 Kÿ1

obtained by averaging all the individual a values wasused for subsequent global ®tting attempts. As expected,the �CP value obtained was small and dif®cult to de®ne,so an average value of 0.31 kcal molÿ1 Kÿ1 was used as a®xed �CP for the entire data set. This value is indistin-guishable from the previous results for a related GCN4coiled coil (Thompson et al., 1993). Finally, the thermo-dynamic parameters �H0and T0 were obtained by per-forming a global ®t to the thermal unfolding curves of theindividual peptides at different concentrations using glo-bal values for YN(0), YU, �Cp and a.

Molecular modeling

The crystal structure (O'Shea et al., 1991) of thepeptide derived from the coiled coil of GCN4(pdb2zta.ent) was used as the starting point for ourmodeling as well as the structure of the dimer(Gonzalez et al., 1996) with the Asn at a postion 16replaced by Gln (pdb1zil.ent). The INII model wasbuilt by grafting a methyl group onto either Cg atomof each a position Val and checking for unfavorablevan der Waals clashes using Insight II 97.0 softwarefrom Molecular Simulations (San Diego, CA). The sol-vent accessibility of the side-chains of the a positionvariants were then calculated according to themethods described by Lee & Richards (1971). For thewild-type sequence, VNVV, the average solvent acces-sibility of the side-chains of the a position residueswas 87(�11) % (range 76-99 %) and for the INII var-iant the average was 83(�13) % (range 59-99 %). Ourconservative estimate is that the average a positionside-chain is �85 % buried in the folded dimer. Itshould be noted that the conclusions based on thisestimate would not be affected if we use the individ-

1386 Buried Polar Groups in Leucine Zippers

ual solvent accessibility values for each a positionrather than the average value.

Acknowledgments

We thank members of the Hu and Scholtz labs forhelpful comments and suggestions. This work was sup-ported by grants from the NIH (GM 52483 to J.M.S.), theNSF (MCB-9305403 and MCB-9808474 to J.C.H.) and theRobert A. Welch Foundation BE-1281 to J.M.S.). J.M.S. isan Established Investigator of the American HeartAssociation.

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Edited by C. R. Matthews

(Received 17 February 2000; received in revised form 7 June 2000; accepted 7 June 2000)