Article No. mb981762 J. Mol. Biol. (1998) 279, 959±972

Inter-helical Interactions in the Leucine Zipper CoiledCoil Dimer: pH and Salt Dependence of CouplingEnergy Between Charged Amino Acids

Dmitry Krylov1, Joe Barchi2 and Charles Vinson1*

1Building 37, Room 4D06Laboratory of Biochemistry and2Laboratory of MedicinalChemistry National CancerInstitute, National Institutes ofHealth, Bethesda, MD 20892USA

Present address: D. Krylov, AmeRockville, MD 20855, USA.

Abbreviations used: CD, circular

0022±2836/98/240959±14 $25.00/0

We have investigated the physical nature of the observed couplingenergy (���Gint) between the charged side-chains of the three inter-heli-cal g$e0 (i, i0 � 5) pairs (E$R, E$K, and E$E) in the leucine zippercoiled coil dimer. Circular dichroism (CD) spectroscopy measured thethermal stability of eight proteins derived from the basic region leucinezipper domain of chicken VBP, the mammalian TEF at seven pHs andthree KCl concentrations. Data from these proteins were used to con-struct double mutant alanine thermodynamic cycles and determine coup-ling energies (���Gint) for the three g$e0 pairs. The attractive E$Rcoupling energy of ÿ0.6 kcal molÿ1 at low salt decreases to ÿ0.2 kcalmolÿ1 at high salt. The E$K coupling energy of ÿ0.5 kcal molÿ1 at lowsalt decreases to ÿ0.1 kcal molÿ1 at high salt. The repulsive E$E coup-ling energy of �0.8 kcal molÿ1 at low salt drops to �0.4 at high salt.Reducing the pH to 2.2 halved the attractive coupling energy for theE$R and E$K pairs while abolishing the repulsion of the E$E pair. 13CNMR of a protein selectively labeled with [13Cd]glutamate that containedthree E$R and one R$E pair identi®ed four glutamates shifted up®eld.We suggest that this is due to electronic perturbation of glutamates ininter-helical E$R interactions. Taken together, these data indicate thatthe E$R coupling energy of ÿ0.5 kcal molÿ1 at pH 7.4 and 150 mM KClhas an electrostatic component.

# 1998 Academic Press Limited

Keywords: coiled coil; coupling energy; electrostatic interactions; leucinezipper; thermodynamic cycle

*Corresponding author

Introduction

The leucine zipper coiled coil is a protein dimeri-zation motif consisting of two parallel amphipathica-helices that is found in a variety of structuralproteins (Cohen & Parry 1990), and the B-ZIP andB-HLH-ZIP families of transcription factors (Murreet al., 1989; Vinson et al., 1989). The coiled coildimerization motif has a structural repeat everytwo a-helical turns or seven amino acids. Each ofthe seven amino acid positions (a,b,c,d,e,f,g) hasunique structural properties, which has made thecoiled coil an attractive model for protein design(Alber 1992; Baxevanis & Vinson 1993; Lupas1996).

The a and d positions are typically hydrophobic,lie on the same side of the amphipathic a-helix,

rican Red Cross,

dichroism.

and are critical for the stability of the dimer(Thompson et al., 1993). The e and g positions¯ank the hydrophobic interface and often containcharged amino acids (McLachlan & Stewart 1975),which have been suggested to regulate dimeriza-tion speci®city (Cohen & Parry 1990). 80% of the eand g positions in the leucine zipper of B-ZIP pro-teins are occupied by the four amino acids, E, Q,K, and R (Vinson et al., 1993). These four aminoacids have long hydrophobic side-chains terminat-ing in either a charged group (E, K, R) or a polargroup (Q). Crystal structures of coiled coils, eitheralone (O'Shea et al., 1991), or as component partsof B-ZIP or B-HLH-ZIP transcription factor dimersbound to DNA (Ellenberger et al., 1994; Ferre-D'Amare et al., 1993; Glover & Harrison 1995),show that the e and g position amino acids lieacross the hydrophobic interface with their side-chain methylene groups interacting with the a andd positions that create the hydrophobic core (Alber1992).

# 1998 Academic Press Limited

960 Inter-helical Coiled Coil Coupling Energy

The contribution of the e and g positions to thestability and speci®city of leucine zipper dimeriza-tion has been actively investigated in recent years(Graddis et al., 1993; Krylov et al., 1994; Nicklin &Casari 1991; O'Shea et al., 1992; Vinson et al., 1993;Zhou et al., 1994). Work by Kim's group, examin-ing the amino acid determinants that drive the het-erodimerization of the FOS and JUN zippers,found repulsive glutamate inter-helical interactionsin the FOS homodimers which mapped to the eand g positions. They suggested that this repulsiveforce is what drove the heterodimerizationbetween the FOS and JUN zippers, not attractiveelectrostatic interactions as suggested from theGCN4 crystal structure (O'Shea et al., 1991).

We have used a double mutant thermodynamiccycle (Serrano et al., 1990) to determine the coup-ling energy (���Gint) between amino acids in theg position of one helix and the following e positionon the other helix of a leucine zipper (the g$e0pair; i, i0 � 5 pair; Krylov et al., 1994). We replacedthe g and e position amino acids with alanine anddetermined the stability of the variant proteins. Byexamining proteins containing alanine in either theg, the e, or both positions, we were able to deter-mine the interaction or coupling energy betweenthe two amino acids in the g$e0 pair. Here wepresent revised data for these coupling energies:the E$R and E$K pair have coupling energiesin 150 mM KCl of ÿ0.5(�0.1) kcal molÿ1 andÿ0.3(�0.15) kcal molÿ1, respectively, while theE$E pair was repulsive with a coupling energy of�0.7(�0.2) kcal molÿ1.

To help unravel the physical nature of the ener-getic interaction between the three g$e0 pairs(E$R, E$K, and E$E), we have determined theircoupling energies at seven pH's and three salt con-centrations. The E$E pair repulsive interaction issuppressed by low pH or high salt. The couplingenergy of the E$R and E$K pair is halved at lowpH or high salt concentrations. All coupling ener-gies were augmented in low salt. 13C NMR exper-iments of a protein containing E$R pairsselectively labeled with [13Cd]glutamate identi®edfour glutamates shifted up®eld as is expected ifthey participate in a E$R salt bridge. These resultssuggest that electrostatic interactions contribute tothe attractive E$R and E$K and repulsive E$Ecoupling energies.

Results

Figure 1A presents a schematic diagram of vitte-logenin binding protein (VBP), our host-guestB-ZIP dimer, bound to DNA with the amino acidsin the leucine zipper region identi®ed (Iyer et al.,1991). Amino acids in the g$e0 pairs of the thirdand fourth heptads have been mutated to alanine(Krylov et al., 1994). This allowed us to construct adouble mutant alanine thermodynamic cycle(Serrano et al., 1990) to determine the interaction orcoupling energy (���Gint) between amino acids

in the g position and the following e0 position, theg$e0, or i, i0 � 5 pair (Vinson et al., 1993). Figure 1Bpresents the amino acid sequence of the leucinezipper for the eight proteins, that were used in thisstudy to construct the thermodynamic cycles forthree g$e0 pairs (E$R, E$K, and E$E). Wemutated two heptads instead of only one for tworeasons: (1) to amplify the signal by examining thefour g$e0 pairs which are found in two heptads(two pairs on each side of the coiled coil homodi-mer) and (2) to minimize any effects that a particu-lar heptad sequence may have on our results. Thiswould lead to more generally applicable proteindesign rules (Krylov et al., 1994). Natural B-ZIPleucine zippers contain a high frequency of oppo-sitely charged amino acids in the g$e0 pair with aclear preference for a E in the g position and a K orR in the following e0 position (Vinson et al., 1993),the type of attractive g$e0 pair we are examininghere.

In a previous manuscript (Krylov et al., 1994),we determined the coupling energy (��Gint),renamed here (���Gint) for 16 g$e0 pairs in abuffer of 12.5 mM phosphate, 150 mM KCl, 1 mMDTT, and 0.25 mM EDTA. To gain insight into thephysical nature of the observed coupling energies,we determined the coupling energy for three ofthese pairs at seven pHs and three salt concen-trations. In the course of this work, we repeatedthe calculation of the coupling energy at pH 7.4and 150 mM KCl for all the proteins. These resultsdiffered from what we obtained previously. Wefound that the A �R34 protein used previously indetermining the E$R coupling energy was actu-ally A �R234, containing an additional glutamate toalanine mutation, which caused it to be destabi-lized relative to A �R34. Using the new data forA �R34, we recalculated four coupling energies atpH 7.4 and 150 mM KCl (Table 1). The calculatedE$R coupling energy is ÿ0.5(�0.1) kcal molÿ1, notthe previously reported ÿ1.14 kcal molÿ1.

Figure 2A presents circular dichroism (CD) spec-tra from 200 to 250 nm for four proteins (A �A4,E �A34, A �R34, and E �R34) at 6�C that were used todetermine the E$R coupling energy. Three exper-imental conditions are presented (pH 7.4, 150 mMKCl; pH 7.4, 1.5 M KCl; pH 2.2, 150 mM KCl). Anassumption in a double mutant thermodynamiccycle is that the protein retains the same three-dimensional polypeptide backbone when a particu-lar amino acid is mutated. The CD spectra for thefour proteins are similar suggesting that themutations have no dramatic effect on the confor-mation of the proteins. We attribute the slightdifference in ellipticity between proteins to exper-imental error in determining protein concen-trations. The samples in low pH show 10% lessellipticity than the two other conditions examined.We do not know if the decrease in ellipticity is dueto a loss of a-helical structure or a decrease in sig-nal for an a-helix at low pH. Similar data wereobtained for the remaining four proteins describedin this study (data not shown).

Figure 1. A, Schematic diagram of the B-ZIP domain of VBP bound to DNA. The N-terminus of the protein is to theleft, followed by the a-helical basic region and leucine zipper coiled coil dimerization domain which extend to theend of the native protein sequence. The amino acid sequence of the parental protein, E �R34, is presented in the con-text of the three-dimensional structure of a coiled coil. To the right are the seven unique positions found in a coiledcoil (a,b,c,d,e,f,g). The four potential g$e0 pairs are indicated by lines between the amino acids. The g$e0 pairs inthe third and fourth heptads which are mutated in this study, are denoted on the top of the Figure and the mutatedresidues are denoted by larger letters. Because of the 2-fold symmetry of the dimers, each heptad contains two g$e0pairs. B, The names and amino acid sequence of the eight proteins used in this study are shown. At the top is thesequence of the parental protein E �R34 coiled coil nomenclature grouped into heptads (g,a,b,c,d,e,f). The third andfourth heptads which have been mutated are noted. The amino acids which are changed from this parental sequenceare indicated for the remaining seven proteins. The coiled coil nomenclature is shown at the bottom.

Inter-helical Coiled Coil Coupling Energy 961

The CD monitored thermal denaturation pro®leswere consistent with the two-state unfolding(Figure 2B) allowing for the determination of ther-modynamic stability parameters. At high salt(1.5 M KCl), the proteins have similar ellipticitybut an increase in stability, which could be due inpart to the screening between the two basic regionsof the dimer. The repulsion between basic regionsis observed for the C/EBP homodimer (Krylovet al., 1995) and the FOS-JUN heterodimer (Oliveet al., 1997). In both cases, the deletion of one basicregion produces a dimer that is 0.5 kcal molÿ1

more stable.Table 1 presents the melting temperature (tm),

enthalpy (�H(tm)), and Gibbs free energy at 37�C(�G) for the eight proteins examined under sevenpH and three KCl concentrations. At neutral pHthe most stable protein studied was E �R34. Lower-ing the pH destabilized ®ve proteins: E �R34, E �K34,

A �A4, A �K34, and A �R34 while stabilizing three:E �E34, E �A34, and A �E34.

�Cp of folding was determined from the slopeof the tm versus �H plot for each of the eight pro-teins using the data from the thermal denatura-tions under different pH and salt concentrations(Figure 3). The �Cp values show the general trendthat those proteins which have oppositely chargedg$e0 pairs (E �R34 and E �K34) have higher �Cp

values. �Cp is directly related to the amount ofhydrophobic surface which is buried upon folding.We suggest that the methylene groups of the side-chains participating in a salt bridge, pack acrossthe dimer interface thus increasing the area ofhydrophobic surface buried in a folded state (Paceet al., 1996). We expect the methylene groups in theeight glutamate residues in the four E$E pairs ofE �E34 not to pack across the hydrophobic interface

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Figure 2. A, Far UV circular dichroism (CD) spectra of four proteins (E �R34, E �A34, A �R34, and A �A4) used to deter-mine the coupling energy (���Gint) for the E$R pair using a double mutant alanine thermodynamic cycle. Threeexperimental conditions are presented. Top panel: pH 7.4, 150 mM KCl; middle panel: pH 2.2, 150 mM KCl; bottompanel: pH 7.4, 1.5 M KCl. All samples contained 12.5 mM phosphate, 0.25 mM EDTA, and 1 mM DTT. B, CD thermaldenaturations of the proteins described in A.

Inter-helical Coiled Coil Coupling Energy 963

which explains the observed low �Cp for E �E34

(ÿ0.6(�0.1) kcal molÿ1 �Cÿ1).Figure 4A presents �G versus pH for the eight

proteins in this study. The most dramatic changein stability is seen for E �E34 which contains gluta-mate in both the g and e0 position in both the thirdand fourth heptad of the leucine zipper. E �E34

becomes 8 kcal molÿ1 more stable at low pH.Figure 4B and Table 2 present ��GA$A, the stab-ility for the seven g$e0 pairs relative to the A$Apair containing alanine in both the g and e0 pos-itions, over a range from pH 2.2 to pH 7.8. Themost stable pair at neutral pH is E$R, being1.3(�0.1) kcal molÿ1 more stable than A$A. The

Figure 3. �Cp was determined from the relationship between �H and tm for each of the eight proteins used in thisstudy. The ®lled points were from the pH experiments and the open circles are from the high and low salt data. Thedata were ®t to a straight line and the calculated �Cp values are indicated with the error.

964 Inter-helical Coiled Coil Coupling Energy

E$K pair is 0.9(�0.13) kcal molÿ1 more stablethan the A$A pair. Above pH 6, only the E$Epair is less stable than A$A. The four pairs con-taining a single alanine (E$A, A$E, A$R, andA$K) are more stable than the A$A pair. Thiscannot be explained by a change in a-helical pro-pensity, since alanine is the best a-helix former.X-ray structures reveal that side-chains at the gand e positions lie across the hydrophobic interface(O'Shea et al., 1991). We suggest that the increasein stability comes from the longer side-chain pack-ing across the hydrophobic interface. This is sup-ported by the observation that the two basic aminoacids, which have longer side-chains, are more sta-bilizing then the shorter glutamic acid in the e pos-ition.

Three g$e0 pairs become more stable at low pH:A$E, E$A, and E$E. The A$E and E$A pairsbecome roughly 0.6 kcal molÿ1 more stable at lowpH. The E$E pair is the most sensitive to pH,changing by 2.0 kcal molÿ1 from a repulsive�0.4(�0.15) kcal molÿ1 at pH 7.4 to an attractiveÿ1.6(�0.13) kcal molÿ1 at pH 2.2. The remaining®ve pairs do not show much change in stabilitywith pH.

Using the data from these eight proteins, wehave constructed a double mutant alanine thermo-dynamic cycle (Figure 5) and determined couplingenergies, ���Gint, for three g$e0 pairs, E$E,E$K, and E$R at each of the seven pHsexamined (Figure 6, Table 3). Alanine is used torepresent a truncated amino acid. The thermodyn-amic cycle determines the energetic interactionbetween two amino acid side-chains to proteinstability (Serrano et al., 1990). E$E shows astrong pH dependence. The coupling energy of�0.7(�0.2) kcal molÿ1 at pH 7.8 drops to

ÿ0.1(�0.2) kcal molÿ1 at pH 2.2. In contrast,the coupling energies for E$R and E$K showa modest pH effect. The E$R couplingenergy decreases from ÿ0.5(�0.1) kcal molÿ1 toÿ0.3(�0.1) kcal molÿ1 and the E$K couplingenergy decreases from ÿ0.3(�0.15) kcal molÿ1 toÿ0.2(�0.15) kcal molÿ1 from neutral to low pH.Graphically, the magnitude of the coupling energyfor the E$R pair can be seen in Figure 4 by addingthe E$A and A$R curves together and compar-ing them to the E$R curve. The larger E$R coup-ling energy compared to E$K can be seen by thelarger difference between the E$R and E$K com-pared to the A$R and A$K curves.

The effect of varying salt concentrations on thestability of the eight proteins in this study wasexamined (Figure 7, Table 2). All proteins werestabilized by the addition of KCl. The proteinsE �R34, E �K34, and A �A4 were 1.0 kcal molÿ1 morestable in 1.5 M KCl than at low salt concentration.E �A34 was stabilized by 1.3 kcal molÿ1. Proteinscontaining a charged amino acid in the e position(A �R34, A �K34, A �E34, ) were stabilized twice asmuch by salt (2.0 to 2.3 kcal molÿ1). The mostsensitive protein was E �E34, being stabilized by4.0(�0.4) kcal molÿ1. Stabilities of the E$R andE$K pairs relative to A$A are not affected bychanges in salt concentration, they remain ÿ1.3and ÿ0.9 kcal molÿ1, respectively.

Coupling energies for E$R and E$K at low salt(12.5 mM phosphate, pH 7.4 and 5 mM KCl) areÿ0.6(�0.1) kcal molÿ1 and ÿ0.5(�0.2) kcal molÿ1

respectively (Figure 8, Table 3). In high salt,(12.5 mM phosphate, pH 7.4 and 1.5 M KCl),the E$R coupling energy is reduced toÿ0.3(�0.1) kcal molÿ1, while the E$K couplingenergy is reduced to ÿ0.1(�0.2) kcal molÿ1. At

ease with which leucine zippers change their oligo-merization properties highlights this issue(Fairman et al., 1996; Harbury et al., 1993; Krylov

Figure 4. A, Stability (�G) of eight proteins at sevenpHs. B, Stability of seven g$e0 pairs relative to theA$A pair (��GA$A) at seven pHs: E$E (®lled dia-monds), E$A (open diamonds), A$E (open down tri-angles), A$K (open up triangles), A$R (open circles),E$K (®lled squares), and E$R (®lled circles). We deter-mined the coupling energy (���Gint) for the threeg$e0 pairs represented by the closed symbols (E$E,E$K, and E$R).

Table 2. Salt and pH effects on the stability of g$e0pairs relative to the A$A pair (��GA$A) (kcal molÿ1)

#g/e0! A E Q R K

pH 7.4, 150 mM KClA 0.0 ÿ0.2 ÿ0.8 ÿ0.7* ÿ0.5E ÿ0.1 �0.4 ÿ0.7 ÿ1.3* ÿ0.9Q ÿ0.4 ÿ0.5 ÿ1.2 ÿ0.8 ÿ0.8R ÿ0.2 ÿ1.6 ÿ0.6 ÿ0.1 ÿ0.1K ÿ0.2 ÿ1.4 ÿ0.7 ÿ0.3 ÿ0.3D > � 1.6 ± ± �0.4 �0.6

pH 2.2, 150 mM KClA 0.0 ÿ0.9 ÿ0.2 ÿ0.3E ÿ0.6 ÿ1.6 ÿ1.1 ÿ1.0

pH 7.4, 1.5 M KClA 0.0 ÿ0.4 ÿ0.8 ÿ0.7E ÿ0.1 0.0 ÿ1.1 ÿ0.9

Values were calculated from Table 1. The error is approxi-mately 0.1 kcal molÿ1. Asterisks marks the values changedfrom Krylov et al. (1994).

Figure 5. Double mutant alanine thermodynamic cycleused to determine coupling energy (���Gint) for theinteraction of glutamic acid in the g position with argi-nine in the following e0 position. The four proteins inthe cycle are A �A4, E �A34, A �R34, and E �R34. The ��Gvalues presented are in terms of an individual g$e0interaction. The E$R pair is 1.26 kcal molÿ1 more stablethan A$A pair. The contribution of the individualamino acids to the stability of the leucine zipper wasdetermined by studying E �A34 and A �R34. The E$Apair is 0.11 kcal molÿ1 more stable than A$A. TheA$R pair is 0.67 kcal molÿ1 more stable than A$A.The sum of the individual contributions of E and R tothe dimer stability is ÿ0.78 kcal molÿ1. The extraÿ0.46 kcal molÿ1 of stability (ÿ1.26-(ÿ0.78)) from theE$R pair is the coupling energy (���Gint) indicativeof the interaction of E with R across the leucine zipper.

Inter-helical Coiled Coil Coupling Energy 965

low salt, the E$E pair is strongly repulsive(�0.8(�0.2) kcal molÿ1). This repulsion decreasesto �0.4(�0.2) kcal molÿ1 at 1.5 M KCl. Both attrac-tive and repulsive interactions can be suppressedby high ionic strength.

To determine a coupling energy, it is critical toknow that under the experimental conditions used,the four proteins that compose the thermodynamiccycle have the same oligomerization states. The

Figure 6. The pH dependence (2.2 to 7.8) of the couplingenergy (���Gint) for three g$e0 pairs E$R (®lled cir-cles), E$K (®lled squares), and E$Eint (®lled dia-monds). We calculated a coupling energy, using adouble mutant thermodynamic cycle, from values inTable 1.

Figure 7. Stability of seven g$e0 pairs relative to theA$A pair (��GA$A) at three salt concentrations: E$E(®lled diamonds), E$A (open diamonds), A$E (opendown triangles), A$K (open up triangles), A$R (opencircles), E$K (®lled squares), and E$R (®lled circles).

966 Inter-helical Coiled Coil Coupling Energy

et al., 1994). We used analytical ultracentrifugationto determine the oligomerization states of proteinsunder different experimental conditions. Allmutants are dimers at pH 7.4 and 150 mM KCl(Krylov et al., 1994). E �K34, E �A34, and E �R34 atpH 2.2 behaved as dimers with calculated molecu-lar weights of 21,000 daltons (data not shown).E �K34, A �R34, and E �R34 at 1.5 M KCl also behavedas dimers (data not shown).

NMR studies were undertaken to determine theelectronic environment at different pHs of the 11glutamate residues found in E �R34, which containsthree E$R pairs and one R$E pair (Figure 9). Pro-teins were labeled with [13Cd]glutamate. 13C NMRspectra could only be obtained when the protein

Table 3. Salt and pH effects on the coupling energiesfor g$e0 pairs (���Gint) (kcal molÿ1)

#g/e0! E Q R K

pH 7.4, 150 mM KClE �0.7 � 0.2 �0.2 � 0.1 ÿ0.5 � 0.1* ÿ0.3 � 0.15Q �0.2 � 0.1 0.0 � 0.1 �0.3 � 0.1* �0.3 � 0.1R ÿ1.1 � 0.1 �0.4 � 0.1 �0.8 � 0.1* �0.8 � 0.1K ÿ0.9 � 0.1 �0.3 � 0.1 �0.6 � 0.1* �0.6 � 0.1

pH 2.2, 150 mM KClE ÿ0.1 � 0.2 ÿ0.3 � 0.1 ÿ0.2 � 0.15

pH 7.4, 0 mM KClE �0.8 � 0.2 ÿ0.6 � 0.1 ÿ0.5 � 0.15

pH 7.4, 1.5 M KClE �0.4 � 0.2 ÿ0.3 � 0.1 ÿ0.1 � 0.2

The coupling energy (���Gint) was calculated from valuesgiven in Table 2. Asterisks marks the values are changed frompreviously reporteded (Krylov et al., 1994).

was expressed in a BL21 strain where the gluta-mate synthetase gene (glnA) was disrupted by P1transduction (Miller, 1992). The glutamate synthe-tase gene product converts glutamate (glutamicacid) to glutamine, which is used for a varietyof metabolic processes which dilutes the[13Cd]glutamate signal. E �R34, has 11 glutamateresidues, four are in the N terminus and are pre-sumed to be in a random coil. The remainingseven are in the coiled coil region, a conclusion

Figure 8. The salt dependence of the coupling energy(���Gint) for the three g$e0 pairs: E$R (®lled circles),E$K (®lled squares), and E$E (®lled diamonds) is pre-sented. A double mutant alanine thermodynamic cycleusing values from Table 1 was used for the calculation.The line through the samples is a linear ®t.

Figure 9. 13C NMR spectra of[13Cd]glutamate labeled E �R34 at62.9 MHz measured at different pHvalues. The sequence of the VBPB-ZIP domain is shown, the topline is presumed to be in a randomcoil while the second line is the leu-cine zipper region and assumed tobe in an a-helical structure. At neu-tral pH, the majority of the gluta-mate residues (blue) are near 181ppm as expected but three or fourare shifted up®eld to 178, asexpected if they are in E$R pairs.As the pH is lowered, all the peaksconverge.

Inter-helical Coiled Coil Coupling Energy 967

drawn from the absolute ellipticity at 222 nmwhich indicates that 60% of the protein is a-helical(Krylov et al., 1994). At pH 6.8, there are two clus-ters of peaks, one consists of a series of peaks ataround 181±182 ppm, the normal chemical shiftregion for a charged glutamate. Another cluster isup®eld at 178±179 ppm, the typical chemical shiftposition for glutamine. As the pH is lowered, bothclusters move up®eld. At pH 2, all peaks clusteraround 179 ppm and the pro®le does not change ifthe pH is lowered to pH 1. We assume that the col-lection of peaks that experience a 4 ppm up®eldshift are in the random coil and in the a-helix, butnot participating in g$e0 pairs. We suggest, bydeduction, that the peaks in the 178±179 ppm clus-ter are in the coiled coil. Seven of the 11 glutamateresidues are expected to be a-helical but only fourare in g$e0 pairs. We suggest that the four gluta-mate residues which cluster at 178±179 ppm areinvolved in hydrogen bonds with arginine in theg$e0 pairs. Assignments of the glutamate residueswill be necessary for further analysis.

Discussion

The existence of attractive inter-helical electro-static interactions in the leucine zipper coiled coilstructure has been debated in the recent literature.Two groups have suggested, using a doublemutant thermodynamic cycle, that attractive inter-actions exist (Krylov et al., 1994; Zhou et al., 1994).

Another group (Lumb & Kim 1995, 1996) did notobserve a pKa shift of glutamate in a putative g$e0pair, suggesting that attractive inter-helical electro-static interactions do not stabilize the coiled-coilstructure. Here we address the physical nature ofthe observed coupling energy (���Gint) in a leu-cine zipper coiled coil for the attractive E$R andE$K, and the repulsive E$E inter-helical g$e0(i, i0 � 5) pairs by three methods: the effect of(1) salt, and (2) pH on a double mutant alaninethermodynamic cycle, and (3) 13C NMR spec-troscopy of [13Cd]glutamate labeled E �R34, adimeric protein containing pairs of three E$R andone R$E g$e0 interactions.

A double mutant alanine thermodynamic cycleanalysis of the g$e0 interaction indicates that anacidic glutamate in the g >position interacts with abasic arginine (E$R) or lysine (E$K) in thefollowing e0 position on the opposite helix withcoupling energies of ÿ0.5 or ÿ0.3 kcal molÿ1,respectively, at neutral pH and 150 mM KCl(Krylov et al., 1994). The coupling energy isincreased in low salt and decreased in high salt.The coupling energies for the attractive E$R andE$K, and repulsive E$E interactions converge tozero in high salts. This convergence indicates thevalidity of the thermodynamic cycle used to con-clude that there is an electrostatic contribution tothe coupling energies. 13C NMR identi®ed fourglutamate residues in the E �R34 protein that wereshifted up®eld, suggesting that these were partially

968 Inter-helical Coiled Coil Coupling Energy

hydrogen bonded and resonated at a chemical shiftsimilar to glutamine as is expected for attractiveE$R and R$E pairs.

Assumptions of a double mutant alaninethermodynamic cycle

The validity of a double mutant thermodynamiccycle to explore the interaction between aminoacids rests on several assumptions (Serrano et al.,1990). First, we assume that the unstructured stateis identical for all four proteins and the residues ofinterest do not interact. Second, the overall fold ofthe polypeptide backbone for the four proteins thatde®ne the cycle are assumed to be the same. Thesimilarity in the oligomerization state and CDellipticities of the mutants suggested that the modi-®cations do not signi®cantly affect the overallstructure. A third assumption is that the chargedamino acid in the alanine g$e0 pair does not par-ticipate in new interactions. However, we knowthat attractive i, i � 3 and i, i � 4 intra-helicalcharged interactions have been identi®ed(Padmanabhan & Baldwin 1994; Scholtz et al.,1993; Szilak et al., 1997). We used the VBP zipperbecause of the limited number of charged aminoacids in the b, c, and f, positions, in an attempt toavoid potential intra-helical charged-charged inter-actions.

Stability versus coupling energy of attractiveg$e0 pairs

An apparent paradox is observed. The E$R pairis 1.3 kcal molÿ1 more stable than an A$A pairand is not destabilized in the presence of high salt(Figure 7). However, the calculated couplingenergy for E$R is salt dependent decreasing fromÿ0.6 kcal molÿ1 in low salt to ÿ0.2 kcal molÿ1 in1.5 M KCl. Examination of the behavior of theA$R and A$K pairs in the thermodynamic cyclehelps to understand this apparent contradiction.Unlike E$R, the A$R and A$K pairs werestabilized by high salt, a result reported earlier (Yuet al., 1996) and also found for monomeric a-helices(Scholtz et al., 1993). Since high salt does not affectthe stability of E$R, but increases the stability ofA$R relative to A$A, the calculated E$R coup-ling energy decreases at high salt. A possiblephysical explanation of these results is that theE$R pair interacts electrostatically and lies acrossthe hydrophobic interface, as suggested by X-raystudies. This can explain how the E$R pair elec-trostratic interaction is not dependent on salt con-centration.

The partially buried nature of charged aminoacids in the g and e positions in the leucine zipperis evidenced by the effect of both pH and salt onstability. Stability analysis of glutamate in the g ore position shows that the charged form of glutamicacid in either of these positions destabilizes acoiled-coil relative to the protonated (uncharged)form. The stability of the E$A pair drops from

ÿ0.6 kcal molÿ1 at pH 2.2 to ÿ0.1 kcal molÿ1 atpH 7.4. For the A$E pair the loss of stabilityassociated with ionization of glutamate is evenmore dramatic: ÿ0.9 kcal molÿ1 at pH 2.2decreases to ÿ0.2 kcal molÿ1 at pH 7.4. The degreeof burial directly affects the free energy costs ofdesolvating and constraining a charged amino acid(Hendsch & Tidor, 1994). We attribute the remain-ing coupling energy at low pH to stabilizationfrom singly charged hydrogen bonds between Eo

and R� or K�. Our data suggest that the inter-actions in the singly charged Eo � � �K� and Eo � � �R�g$e0 pairs are weaker than the ion-pair interactionbetween the ionized form of glutamate and argi-nine or lysine, although they still provide consider-able stabilization to the coiled-coil. The strength ofsingly charged hydrogen bonds was previouslymeasured for i, i � 3 and i, i � 4 intra-helical E$Kpairs (Scholtz et al.,1993) and was also found to beweaker than the corresponding ion-pair interaction(�0.3 kcal molÿ1 as opposed to 0.45 kcal molÿ1).

The A$E pair is more stabilized by salt than theE$A pair. A possible explanation lies in a differ-ent degree of solvent exposure of the side-chains inthe e and g positions. Charged side-chains whichare more buried will be more responsive to thechanges in ionic strength. We hypothesize that thecharged glutamate in the e position is less solvent-exposed than in the g position and therefore moreresponsive to the changes in salt concentration.This is con®rmed by model building: the e positionside-chain points into the inter-helical hydrophobicinterface allowing the side-chain methylene groupsto lie across the interface and still participate in theg$e0 interactions. The g position side-chain ispointed away from the interface into solution.

Attractive versus repulsive inter-helical interactions

Several groups have examined the contributionof attractive charged interactions between a-helicesin model dimeric (Vinson et al., 1993; O'Shea et al.,1993; Krylov et al., 1994; Zhou et al., 1994; Lumb &Kim 1995, 1996; Szilak et al., 1997) and tetrameric(Fairman et al., 1996) coiled coil structures. A num-ber of studies have shown that a mixture of acidicand basic leucine zippers is more stable than eitheralone (Fairman et al., 1996; Graddis et al., 1993;Krylov et al., 1994; O'Shea et al., 1992). This type ofdata has been taken to suggest that there is anattractive electrostatic interaction between theoppositely charged amino acids on the two helices.An alternative explanation is that repulsive inter-actions between similarly charged side-chains dis-courage the formation of homodimers thusfavoring heterodimerization. This interpretation issupported by the absence of a pH dependence forthe formation of the FOS-JUN heterodimer (O'Sheaet al., 1992).

Our analysis indicates that in mixtures of acidicleucine zippers, containing E$E pairs, and basicleucine zippers, containing R$R pairs, the speci-

Inter-helical Coiled Coil Coupling Energy 969

®city of dimerization is determined by repulsion inhomodimers and attraction in heterodimers. TheE$E and R$R pairs both have a repulsive coup-ling energy of �0.7 kcal molÿ1. The heterodimerwould have attractive E$R and R$E pairs withcoupling energies of ÿ0.5 kcal molÿ1 and ÿ1.0 kcalmolÿ1, respectively. These data suggest that boththe repulsion of the homodimers and the attractionof the heterodimers are critical for heterodimer for-mation.

Intra-helical versus inter-helicalattractive interactions

The contribution of electrostatic interactions toprotein stability has been actively addressed inrecent years (Hendsch & Tidor, 1994; Yang &Honig, 1992). The coupling energy (���Gint)between oppositely charged amino acids in theinterior of proteins can be ÿ3 to ÿ5 kcal molÿ1

(Anderson et al., 1990), while on the surface ofproteins, electrostatic interactions contributeÿ0.5 kcal molÿ1 to protein stability. Intra-helicalelectrostatic interactions between charged aminoacids on the surface of a-helices in native proteinsand in model a-helical peptides have been investi-gated. In the protein barnase, only ÿ0.2 kcal molÿ1

of coupling energy was observed for a i, i � 4intra-helical salt bridge between glutamate andlysine (Sali et al., 1991). Others, using a monomerica-helical peptide system, have identifed aÿ0.5 kcal molÿ1 interaction energy for the i, i � 4intra-helical salt bridge between glutamate andlysine (Lyu et al., 1992; Scholtz et al., 1993).

Baldwin's group has produced a data set forintra-helical i, i � 3 and i, i � 4 interactionsbetween glutamate and lysine using a doublemutant alanine thermodynamic cycle at differentpH and salt conditions (Scholtz et al., 1993). Themagnitude of the calculated intra-helical couplingenergies at different pHs and salts were similar tothose we observe for inter-helical interactions(Figure 7).

Examining the four attractive i, i � 4 intra-helicalinteractions, E-R, E-K, D-K and D-R, investigatorsconcluded that R is more stabilizing than K, and Eis more stabilizing than D (Huyghues-Despointeset al., 1993; Merutka & Stellwagen 1991). This is thesame order of stabilities we observe for g$e0 pairs.The difference in energetic contribution betweenthe two acidic amino acids is 1.5 kcal molÿ1

presumably because the short D side-chain placesthe acidic group too close to the hydrophobic inter-face. The difference between R and K is only0.2 kcal molÿ1.

Correlation of NMR data and couplingenergy calculations

The NMR data qualitatively support the con-clusions of the double mutant alanine thermodyn-amic cycle. We suggest that the measuredattractive coupling energy for the E$R pair is

re¯ected in the up®eld shift seen in the 13C NMRsignal for [13Cd]glutamate labeled protein. We havepresented 13C NMR data that indicate that[13Cd]glutamate labeled E �R34 protein contains fourglutamate residues that are shifted 2 ppm up®eldtoward the position of glutamine at neutral pH.This is expected if glutamate residues are interact-ing favorably with arginine in a g$e0 pair.Additional experiments will be required to unam-biguously assign the glutamate residues and con-®rm that the up®eld shifted peaks are fromglutamate in E$R pairs.

Using 13C NMR of [13Cd]glutamate, Kim's groupdid not observe a change in pKa or ppm upon fold-ing for glutamate residues in either an E$K(Lumb & Kim 1995) or E$R (Lumb & Kim, 1996)pair that were observed to interact inter-helically inthe GCN4 zipper crystal structure (O'Shea, et al.,1991). The presence of g$e0 attractive chargedinteractions in the VBP zipper and their absence inthe GCN4 zipper highlight our limited understand-ing of this subtle and complex problem. Severalpossibilities exist to explain the difference in the13C NMR data between the two model systems.A possibility is that the three sequential E$R pairsalong the coiled coil in our model system may actcooperatively to enhance both the measured coup-ling energies and the diminished ppm shift. Alter-natively, the VBP zipper was covalently closed at acysteine in the d position which may stabilize theelectrostatic interactions of the g$e0 pairs.

Experimental Procedures

Proteins

The names of the eight proteins used in this study arein Table 1. For example in E �R34, the large dot denotesthat this a protein name, the E and R are the amino acidsin the g and e position of the g$e0 pair, and the sub-script numbers identify the heptad with this g$e0 pair.The sequence of the 96 amino acid host protein, E �R34, isASMTGGQQMGRDP-LEE-KVFVPDEQKDEKYWTRRK-KNNVAAKRSRDARRLKENQ ITIRAAF LEKENTALRTEVAE LRKEVGR CRNIVSK YETRYGPL. The ®rst16 amino acids are from f10 (Studier & Moffatt, 1986)and a cloning linker, and the remaining 80 amino acidsare the entire C-terminus of VBP (Iyer et al., 1991). These80 amino acids contain the B-ZIP domain, which iscapable of sequence-speci®c DNA binding. The leucinezipper region is blocked into heptads (d,e,f,g,a,b,c). Thed positions are in bold type. The invariant asparagineand arginine of the basic region are italicized. All themutant proteins were described previously (Krylov et al.,1994).

Protein expression and purification

Proteins were expressed in Escherichia coli using thephage T7 expression system (Studier & Moffatt 1986),and puri®ed as described earlier (Krylov et al., 1994).Proteins puri®ed from the heparin column were sub-sequently chromatographed to homogeneity, using a C18

column on a Rainin HPLC system, with a 0 to 100%acetonitrile gradient in 0.1% (w/v) tri¯uoroacetic acid.

970 Inter-helical Coiled Coil Coupling Energy

All protein concentrations were determined by absor-bance at 280 nm using a Hewlett Packard 8425A spectro-photometer, assuming the known absorbance for the onetryptophan and four tyrosine residues in the molecule(e280 � 10,000; Cantor & Schimmel, 1980). Absorbance at280 nm in either the circular dichroism buffer or 6 Mguanidine hydrochloride were identical.

13C labeling of proteins

[13Cd]glutamate labeling of the E �R34 protein was car-ried out using the E. coli strain BL21 which was modi®edto disrupt the metabolic conversion of glutamate to glu-tamine. This was accomplished by disrupting the gluta-mate synthetase gene (glnA) by P1 transduction (Miller,1992). The glnA gene was marked with tetracyclineresistance (zig-219::Tn10) from a strain designated CGSCStrain no. 6175, which was obtained from the Yale E. colistock center. Proteins were grown in de®ned amino acidmedium (Muchmore et al., 1989). A 10 ml overnight cul-ture containing 100 mM glutamate was added to a400 ml culture containing 100 mM [13Cd]glutamate. Themutant strain was essential to produce a 13C NMR sig-nal.

Circular dichroism

Circular dichroism (CD) studies were performed usinga Jasco J-720 spectropolarimeter with a 5-mm rectangularCD cell. All protein stock solutions were in 12.5 mM pot-assium phosphate (pH 7.4), 150 mM KCl, 1 mM EDTA,1 mM DTT (CD buffer). DTT was added to a 1.15 � 10ÿ4

M 50 ml sample in the above buffer, heated to 65�C for15 minutes to disrupt a potential disul®de bond betweentwo cysteine residues in the ®fth d position, cooled toroom temperature for ®ve minutes and then diluted to3.4 mM in a 1.7 ml sample volume in the appropriatebuffer. The low pH samples were prepared as follows:protein solutions with de®ned pHs were prepared bydiluting 50 ml of protein stock solution into 1.650 ml of12.5 mM potassium phosphate buffer with the pH ran-ging from 2 to 7.8. Buffers with different pH were pre-pared by mixing 12.5 mM H3PO4, 12.5 mM KH2PO4 andK2HPO4 in different proportions. pH of the protein sol-ution was measured by a Orion pH-meter equippedwith NMR-tube electrode ``Ingold''.

Thermodynamic calculations

Melting temperature (tm) and enthalpy (�H) valueswere determined from denaturation curves assuming atwo-state equilibrium dissociation of a-helical dimersinto unfolded monomers using the following equation:

y�t� � f�yM ÿ yD���1ÿ t=tint���1� �1ÿ SQRT�8�EXP��1=R���H��1=�tm � 273�ÿ 1=�t� 273��� � 1��=�4�EXP��1=R���H��1=�tm

� 273� ÿ 1=�t� 273����� � yDg;where y(t) is the ellipticity of the sample as a function oftemperature (t); yD is the ellipticity of the dimer att � 0�C; yM is the ellipticity of the monomer; tint is thetemperature at which yD, assumed to be a linear functionof temperature, becomes equal to yM; �H is van't Hoffenthalpy of the transition; and tm is the melting tempera-ture (Krylov et al., 1994). �Cp were obtained from therelationship between tm versus �H for each protein atvarious pH and salt conditions reported. �G values

were obtained from tm, �H, and �Cp. Using a single�Cp value averaged from all eight proteins (ÿ1.2 kcalmolÿ1), the coupling energy calculations were within�0.15 kcal molÿ1 of the values obtained using individual�Cp values. �G values are reported at 37�C. The differ-ence in thermal stability between the two proteins X �Y34,where X and Y represent any amino acid, and E �R34

re¯ects the substitution of four E$R pairs with fourX$Y pairs, and therefore, the stability of X$Y pairrelative to the E$R pair is given by��GX$Y � (�G(X �Y34)-�G(E �R34))/4. Unlike othermutants, A �A4 has only one A$A pair substituted forE$R. The stability of the A$A pair relative to E �R34 isequal to ��GA$A � ��G�A �A4�-�G(E �R34))/2. Thisstability was subtracted from the stabilities of all otherpairs to produce a set of stabilities relative to the A$Apair, which we denote as ��GA$A. For example,the interaction or coupling energy (���Gint) calcu-lations for E$R pairs were calculated using��GA$A � 0 kcal molÿ1, ��GE$A, ��GA$R, and��GE$R. Previously, we called the coupling energy,��Gint (Krylov et al., 1994). All thermal melts werereversible. The A �R34 melts at low pH did not have aclear initial base line, which complicates the curve ®tting.To overcome this dif®culty, we assumed the initial baseline to be similar to the other proteins. After this initialassumption, the base line was not constrained in the ®t-ting procedure.

The standard errors for tm and �H determined fromthe ®tting procedure were typically not higher than0.4�C and 0.5 kcal/mol, respectively. These errors alongwith the �Cp error were individually translated into theerror in �G by substituting the extreme tm, �H and �Cp

values in the equation for �G. These individual errorswere added up to produce an overall error for �G, pre-sented in Table 1. The errors for coupling energies aregenerally given by the sum of the errors in free energyfor the proteins comprising the double mutant cycle. Inour case this error was divided by 4 because the coup-ling energy was averaged for four interacting g$e0pairs.

Equilibrium sedimentation

Equilibrium sedimentation measurements were per-formed using a Beckman XL-A Optima Analytical Ultra-centrifuge equipped with absorbance optics and aBeckman An-60Ti rotor. Samples were loaded at threeconcentrations, 10, 20, and 40 mM (0.1, 0.2 and 0.4 A at280 nm), into a six well centerpiece and spun at 6�C and25,000 rpm for 24 hours. 20 data sets for three concen-trations were jointly ®t for a singular molecular mass.Some calculations assumed a monomer-dimer equili-brium. Compositional partial speci®c volumes for theproteins were calculated as described by Zamyatnin(1984).

NMR spectra

13C NMR spectra were recorded on a Bruker AC250spectrometer at a carbon frequency of 62.9 MHz using a1H/13C switchable probe. Broadband 1H decoupling wasperformed with WALTZ16 (Shaka et al., 1983) compositepulses. One-dimensional spectra were obtained with aspectral width of 14,705.8 Hz and a recycle delay of onesecond. Spectra were referenced to internal dioxane-d8

set at 66.5 ppm. Datasets were collected with 32,000 data

Inter-helical Coiled Coil Coupling Energy 971

points and the number of scans per experiment was typi-cally between 15,000 and 20,000.

Acknowledgment

We thank Susan Gottesman, Susan Garges andDhruba Chattoraj for help with the P1 transduction ofBL21 to create the mutant in glutamate utilization, B.K.Lee for insightful conversations, and Valda Vinson forcomments on the manuscript.

References

Alber, T. (1992). Structure of the leucine zipper. Curr.Opin. Genet. Dev. 2, 205±210.

Anderson, D., Becktel, W. & Dahlquist, F. (1990). pH-induced Denaturation of proteins: a single saltbridge contributes 3-5 kcal/mol to the free energyof folding of T4 lysozyme. Biochemistry, 29, 2403±2408.

Baxevanis, A. & Vinson, C. (1993). Interactions of coiledcoils in transcription factors: where is thespeci®city?. Curr. Opin. Genet. Dev. 3, 278±285.

Cantor, C. & Schimmel, P. (1980). Biophysical Chemistry,W. H. Freeman, New York.

Cohen, C. & Parry, D. (1990). A-helical coiled coils andbundles: how to design an a-helical protein. Protein:Struct. Funct. Genet. 7, 1±14.

Ellenberger, T., Fass, D., Arnaud, M. & Harrison, S.(1994). Crystal structure of transcription factor E47:e-box recognition by a basic helix-loop-helix dimer.Genes Dev. 8, 970±980.

Fairman, R., Chao, H. G., Lavoie, T. B., Villafranca, J. J.,Matsueda, G. R. & Novotny, J. (1996). Design ofheterotetrameric coiled coils: evidence for increasedstabilization by Glu(ÿ)-Lys(�) ion pair interactions.Biochemistry, 35, 2824±2829.

Ferre-D'Amare, A., Prendergast, G., Ziff, E. & Burley, S.(1993). Recognition by Max of its cognate DNAthrough a dimeric b/HLH/Z domain. Nature, 363,38±44.

Glover, M. & Harrison, S. (1995). Crystal structure ofthe heterodimeric B-ZIP transcription factor c-Fos±c-Jun bound to DNA. Nature, 373, 257±261.

Graddis, T., Myszka, D. & Chaiken, I. (1993). Controlledformation of model homo- and heterodimer coiledcoil polypeptides. Biochemistry, 32, 12664±12671.

Harbury, P., Zhang, T., Kim, P. & Abler, T. (1993).A switch between two-, three-, and four-strandedcoiled coils in GCN4 leucine zipper mutants.Science, 262, 1401±1407.

Hendsch, Z. & Tidor, B. (1994). Do salt bridges stabilizeproteins?A continuum electrostatic analysis. ProteinSci. 3, 211±226.

Huyghues-Despointes, B., Scoltz, J. & Baldwin, R.(1993). Helical peptides with three pairs of Asp-Argand Glu-Arg residues in different orientations andspacings. Protein Sci. 2, 80±85.

Iyer, S., Davis, D., Seal, S. & Burch, J. (1991). Chickenvitellogenin gene- binding protein, a leucine zippertranscription factor that binds to an important con-trol element in the chicken vitellogenin II promoter,is related to rat DBP. Mol. Cell. Biol. 11, 4863±4875.

Krylov, D., Mikhailenko, I. & Vinson, C. (1994). A ther-modynamic scale for leucine zipper stability and

dimerization speci®city: e and g inter-helicalinteractions. EMBO J. 13, 1849±1861.

Krylov, D., Olive, M. & Vinson, C. (1995). Extendingdimerization interfaces: the B-ZIP basic region canform a coiled coil. EMBO J. 14, 5329±5337.

Lumb, K. & Kim, P. (1995). Measurement of inter-helicalelectrostatic interactions in the GCN4 leucinezipper. Science, 268, 436±439.

Lumb, K. & Kim, P. (1996). Interhelical salt bridges,coiled-coil stability and speci®city of dimerization(technical comment). Science, 271, 1137±1138.

Lupas, A. (1996). Coiled coils: new structures and newfunctions. Trends Biochem. 21, 375±382.

Lyu, P. C., Gans, P. J. & Kallenbach, N. R. (1992).Energetic contribution of solvent-exposed ion pairsto alpha-helix structure. J. Mol. Biol. 223, 343±350.

McLachlan, A. & Stewart, M. (1975). Tropomyosincoiled-coil interactions: evidence for an unstaggeredstructure. J. Mol. Biol. 98, 293±304.

Merutka, G. & Stellwagen, E. (1991). Effect of aminoacid ion pairs on peptide helicity. Biochemistry, 30,1591±1594.

Miller, J. H. (1992). A Short Course in Bacterial Genetics: ALaboratory Manual and Handbook for Escherichia Coliand Related Bacteria, pp. 263±277, Cold Spring Har-bor Laboratory Press, Cold Spring Harbor, NY.

Muchmore, D., McIntosh, L., Anderson, C. R. D. &Dahlquist, F. (1989). Expression and nitrogen-15labeling of proteins for proton and nitrogen-15nuclear magnetic resonance. Methods Enzymol. 177,44±73.

Murre, C., McCaw, P. & Baltimore, D. (1989). A newDNA binding and dimerization motif in immuno-globulin enhancer binding, daughterless, MyoD,and myc proteins. Cell, 56, 777±783.

Nicklin, M. & Casari, G. (1991). A single site mutationin a truncated Fos protein allows it to interact withthe TRE in vitro. Oncogene, 6, 173±179.

O'Shea, E., Klemm, J., Kim, P. & Abler, T. (1991). X-raystructure of the GCN4 leucine zipper, a two-stranded, parallel, coiled-coil. Science, 254, 539±544.

O'Shea, E., Rutkowski, R. & Kim, P. (1992). Mechanismof speci®city in the fos-jun oncoprotein heterodimer.Cell, 68, 699±708.

O'Shea, E., Lumb, K. & Kim, P. (1993). Peptide velcro:design of a heterodimeric coiled coil. Curr. Biol. 3,658±667.

Olive, M., Krylov, D., Echlin, D. R., Gardner, K.,Taparowsky, E. & Vinson, C. (1997). A dominant-negative to AP1 that abolishes DNA binding andinhibits oncogenesis. J. Biol. Chem. 272, 18586±18594.

Pace, C. N., Shirley, B. A., McNutt, M. & Gajiwala, K.(1996). Forces contributing to the conformationalstability of proteins. FASEB J. 10, 75±83.

Padmanabhan, S. & Baldwin, R. (1994). Tests of helix-stabilizing interactions between various nonpolarside-chains in alanine-based peptides. Protein Sci. 3,1992±1997.

Sali, D., Bycroft, M. & Fersht, A. (1991). Surface electro-static interactions contribute little to the stability ofbarnase. J. Mol. Biol. 220, 779±788.

Scholtz, J., Qian, H., Robbins, V. & Baldwin, R. (1993).The energetics of ion-pair and hydrogen-bondinginteractions in a helical peptide. Biochemistry, 32,9668±9676.

Serrano, L., Horovitz, A., Avron, B., Bycroft, M. &Fersht, A. (1990). Estimating the contribution ofengineering surface electrostatic interactions to pro-

972 Inter-helical Coiled Coil Coupling Energy

tein stabvility using double mutant cycles. Biochem-istry, 29, 9343±9352.

Shaka, A., Keller, R. & Freeman, R. (1983). Evaluation ofa new broadband decoupling sequence: WALTZ-16.J. Magn. Reson. 53, 313±340.

Studier, F. W. & Moffatt, B. A. (1986). Use of bacterio-phage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189,113±130.

Szilak, L., Moitra, J. & Vinson, C. (1997). Design of aleucine zipper coiled coil stabilized 1.4 kcal molÿ1

by phosphorylation of a serine in the e position.Protein Sci. 6, 1273±1283.

Thompson, K., Vinson, C. & Freire, E. (1993).Thermodynamic characterization of the structuralstability of the coiled-coil region of the B-ZIP tran-scription factor GCN4. Biochemistry, 32, 5491±5496.

Vinson, C. R., Sigler, P. B. & McKnight, S. L. (1989).A scissors-grip model for DNA recognition by a

family of leucine zipper proteins. Science, 246, 911±916.

Vinson, C. R., Hai, T. W. & Boyd, S. M. (1993).Dimerization speci®cty of the leucine zipper-con-taining B-ZIP motif on DNA binding: predictionand rational design. Genes Dev. 7, 1047±1058.

Yang, A. & Honig, B. (1992). Electrostatic effects on pro-tein stability. Curr. Opin. Struct. Biol. 2, 40±45.

Yu, Y., Monera, O., Hodges, R. & Privalov, P. (1996).Investigation of electrostatic interactions in two-stranded coiled-coils through residue shuf¯ing. Bio-phys. Chem. 59, 299±314.

Zamyatnin, A. (1984). Amino acid, peptide, and proteinvolume in solution. Annu. Rev. Biophys. Bioeng. 13,145±165.

Zhou, N., Kay, C. & Hodges, R. (1994). The net ener-getic contribution of inter-helical electrostatic attrac-tions to coiled-coil stability. Protein Eng. 7, 1365±1372.

Edited by M. F. Moody

(Received 16 June 1997; received in revised form 4 March 1998; accepted 6 March 1998)