Aspartic acid 50 and tyrosine 108 are essential for receptor binding and cytotoxic activity of tumour necrosis factor beta (lymphotoxin)

Single amino acid substitutions were generated in predictedhydrophilic loop regions of the human tumour necrosis factorbeta (TNF-ß) molecule, and the mutant proteins wereexpressed in Escherichia coli and purified. Mutants with singleamino acid changes at either of two distinct loop regions, atpositions aspartic acid 50 or tyrosine 108, were found to havegreatly reduced receptor binding and cytotoxic activity. Thesetwo regions in TNF-ß correspond to known loop regionswhere mutations also result in loss of biological activity ofTNF–, a related cytokine which shares the same cellularreceptors with TNF-ß. The two distinct loops at positions31-34 and 84-89 in the known three-dimensional structure ofTNF- (equivalent to positions 46–50 and 105–110respectively in TNF-ß), lie on opposite sides of theTNF- monomer. When the TNF-a monomer forms a trimer, the twoloops, each from a different subunit of the trimer, come togetherand lie in a cleft between adjacent subunits. Together, thesefindings suggest that a TNF receptor binds to a cleft betweensubunits via surface loops at amino acid residues 31–34and 84–89 in TNF–, and similarly via surface loopsincluding amino acids aspartic acid 50 and tyrosine 108 in TNF–ß.     

Characterization of the apparent negative co-operativity induced in Escherichia coli aspartate aminotransferase by the replacement of Asp222 with alanine. Evidence for an extremely slow conformational change

The strictly conserved active site residue, Asp222, which formsa hydrogen-bonded salt bridge with the pyridine nitrogen atomof the pyridoxal 5' phosphate (PLP) co-factor of aspartate aminotransferase(AATase), was replaced with alanine (D222A) in the Escherichiacoli enzyme. The D222A mutant exhibits non–hyberbolicsaturation behavior with amino acid substrates which appearas apparent negative eooperativity in steady–state kineticanalyses. Single turnover progress curves for D222A are welldescribed by the sum of two exponentials, contrasting with themonophasic kinetics of the wild-type enzyme. An active/inactiveheterodimer containing the D222A mutation retains this biphasickinetic response, proving that the observed eooperativity isnot the result of induced allostery. The anomalous behavioris explained by a hysteretic kinetic model involving two slowlyinterconverting enzyme forms, only one of which is catalyticallycompetent. The slow functional transition between the two formshas a half–life of 10 mins. Preincubation of the mutantwith the dicarboxylk inhibitor maleate shifts the equilibriumpopulation of the enzyme towards the catalytically active form,suggesting that the slow transition is related to the domainclosure known to occur upon association of this inhibitor withthe wild-type enzyme. The importance of Asp222 in the chemicalsteps of transamination is confirmed by the l0⁵fold decreasein catalytic competence in the D222A mutant, and by the largeprimary C–deuterium kinetic isotope effect (6.7 versus2.2 for the wild–type). The transamination activity ofthe D222A mutant is enhanced 4– to 20–fold by reconstltutionwith the co-factor analog N–methylpyridoxal–5–phosphate(N–MePLP), and the C–proton abstraction step isless rate determining, as evidenced by the decrease in the primarykinetic isotope effect from 6.7 to 2.3. These results suggestthat the conserved interaction between the protonated pyridinenitrogen of PLP and the negatively charged carboxylate of Asp222is important not only for efficient C–proton abstraction,but also for conformational transitions concomitant with thetransamination process     

Effects of amino acid substitutions in the hydrophobic core of {alpha}-lactalbumin on the stability of the molten globule state

Five mutant –lactalbumins, with one or two amino acidsubstitution(s) in the B helix, were engineered to examine therelation between the stability of the molten globule state andthe hydrophobicity of these amino acids. The mutation sites(Thr29, Ala30 and Thr33) have been chosen on the basis of comparisonof the amino acid sequences of goat, bovine and gunea pig –lactalbumin,in which the guinea pig protein shows a remarkably more stablemolten globule than the other proteins. The recombinant proteinswere expressed Escherichia coli and then purified and refoldedefficiently to produce the active proteins. The stability ofthe molten globule state of these engineered proteins has beeninvestigated by urea–induced unfolding transition underan acidic condition (pH 2.0), where the molten globule stateis stable in the absence of urea. The results show that themolten globule state is stabilized by the amino acid substitutionswhich raise the hydrophobicity of the residues, suggesting thatthe hydrophobic core in a globular protein plays an importantrole in the stability of the molten globule state. The changein stabilization free energy of the molten globule state causedby each amino acid substitution has been evaluated, and molecularmechanisms of stabilization of the molten globule state arediscussed.     

Studies of the pore-forming domain of a voltage-gated potassium channel protein

Recent mutagenesis studies nave identified a stretch of aminoacid residues which form the ion-selective pore of the voltage-gatedpotassium channel. It has been suggested that this sequenceof amino acids forms a ß-barrel structure making upthe structure of the ion-selective pore [Hartman,H.A., Kirsch,G.E.,DreweJ.A., Taglialatela.M., Joho.R.H. and Brown,A.M. (1991)Science, 251, 942–944; YeUen.G., Jurman,M.E., Abramson,T.and MacKinnon,R. (1991) Science, 251, 939–942; Yool,AJ.and Schwarz.T.L. (1991) Nature, 349, 700–704]. We havesynthesized a polypeptide corresponding to this amino add sequence(residues 431–449 of the ShA potassium channel from Drosophila).A tetrameric version of this sequence was also synthesized byUnking together four of these peptldes onto a branching lysinecore. Fourier transform infrared (FT-LR) and circular dichroism(CD) spectroscopy have been used to investigate the structureof these peptides after their reconstitution into lyso phos-phatidylcholinemicelles and lipid bilayers composed of dimyristoyl phosphatidyfcholineand dimyristoyl phosphatidyl-glycerol. The spectroscopic studiesshow that these peptides are predominantly a-helical in theselipid environments. When Incorporated into planar lipid bilayersboth peptides induce ion channel activity. Molecular modellingstudies based upon the propensity of these peptides to forman -helical secondary structure in a hydrophobfc environmentare described. These results are discussed in the light of recentmutagenesis and binding studies of the Drosophila Shaker potassiumion channel protein     

Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling

The possibility of increasing the affinity of a Taq DNA polymerasespecific binding protein (affibody) was investigated by an -helixshuffling strategy. The primary affibody was from a naive combinatoriallibrary of the three-helix bundle Z domain derived from staphylococcalprotein A. A hierarchical library was constructed through selectivere-randomization of six amino acid positions in one of the two-helices of the domain, making up the Taq DNA polymerase bindingsurface. After selections using monovalent phage display technology,second generation variants were identified having affinities(K_D) for Taq DNA polymerase in the range of 30–50 nM asdetermined by biosensor technology. Analysis of binding dataindicated that the increases in affinity were predominantlydue to decreased dissociation rate kinetics. Interestingly,the affinities observed for the second generation Taq DNA polymerasespecific affibodies are of similar strength as the affinitybetween the original protein A domain and the Fc domain of humanimmunoglobulin G. Further, the possibilities of increasing theapparent affinity through multimerization of affibodies wasdemonstrated for a dimeric version of one of the second generationaffibodies, constructed by head-to-tail gene fusion. As comparedwith its monomeric counterpart, the binding to sensor chip immobilizedTaq DNA polymerase was characterized by a threefold higher apparentaffinity, due to slower off-rate kinetics. The results showthat the binding specificity of the protein A domain can bere-directed to an entirely different target, without loss ofbinding strength.     

The predicted secondary structure of the G-type glutamineamidotransferase is compatible with TEM-barrel topology

Glutamine amidotransferase (GAT) subunits or domains catalyzean important partial reaction in many complex biosynthetic reactions.The structure of one member of the F-type GATs is known, butthe structure of the unrelated G-type is still unknown. Becausemany protein sequences are available for anthranilate synthasecomponent II (product of the trpG gene), we have predicted itsaverage secondary structure by a joint prediction method [Niermannand Kirschner (1991a) Protein Engng, 4, 359–370]. Thepredicted eight ß-strands and seven -helices followan 8-fold cyclic repetition of a ß-strand-loop--helix-loopmodule with helix 7 missing. This pattern of secondary structuresuggests that the G-type GAT domain has an 8-fold ß-barreltopology, as found first in triose phosphate isomerase (TIM-barrel).This model is supported by the location of known catalyticallyessential residues in loops between (ß-strands and-helices. Evidence from published sequencing and mutationalstudies on selected members of the GAT superfamily (carbamoylphosphate, imidazoleglycerol phosphate, GMP and CTP synthases)support both the secondary structure prediction and the TIM-barreltopology.     

Characterization of the DNA binding domain of the mouse IRF-2 protein

The DNA binding domain of the interferon regulatory factor-2protein (IRF-2) has been produced and characterized, -chymotrypsindigestion of the purified IRF-2 protein bound to a syntheticbinding site yields a peptide fragment of 14 K in molecularweight. N-terminal analysis of this peptide fragment showedthat its sequence is the same as that of the intact IRF-2. Apeptide fragment of {small tilde} 14 K, IRF-2(113), which correspondsto the N-terminal 113 amino acids of the intact IRF-2 protein,has been expressed in a functional form in Escherichia coli.The first methionine was processed during the expression andthe purified IRF-2(113) thus contains 112 amino acids. DNaseI footprinting and gel retardation assaying showed that IRF-2(113)binds to a synthetic DNA having the consensus binding site andto the upstream regulatory sequence of the IFN-ß geneas intact IRF-2 does. These results showed that this peptidefragment, IRF-2(113), may be a good material for investigationof the DNA binding domain of IRF-2 and of the DNA–proteininteraction.     

The {alpha}/{beta} hydrolase fold

We have identified a new protein fold—the /ßhydrolase fold—that is common to several hydrolytic enzymesof widely differing phylogenetic origin and catalytic function.The core of each enzyme is similar: an /ß sheet, notbarrel, of eight ß-sheets connected by -helices. Theseenzymes have diverged from a common ancestor so as to preservethe arrangement of the catalytic residues, not the binding site.They all have a catalytic triad, the elements of which are borneon loops which are the best-conserved structural features inthe fold. Only the histidine in the nucleophile-histidine-acidcatalytic triad is completely conserved, with the nucleophileand acid loops accommodating more than one type of amino acid.The unique topological and sequence arrangement of the triadresidues produces a catalytic triad which is, in a sense, amirror-image of the serine protease catalytic triad. There arenow four groups of enzymes which contain catalytic triads andwhich are related by convergent evolution towards a stable,useful active site: the eukaryotic serine proteases, the cysteineproteases, subtilisins and the /ß hydrolase fold enzymes.     

A new method for random mutagenesis of complete genes: enzymatic generation of mutant libraries in vitro

A new efficient in vitro mutagenesis method for the generationof complete random mutant libraries, containing all possiblesingle base substitution mutations in a cloned gene is described.The method is based on controlled use of polymerases. Four populationsof DNA molecules are first generated by primer elongation sothat they terminate randomly, but always just before a knowntype of base (before A, C, G or T respectively). Each of thefour populations is then mutagenized in a separate misincorporationreaction, where the correct base can now be omitted. The regenerationof wild-type sequences can thus be efficiently avoided. Also,the misincorporating nucleotide concentrations can be optimizedto give the three possible single mutations in close to equalratio. The mutagenesis can be precisely localized within a predeterminedtarget region of any size, and vector sequences remain intact.We have mutagenized the DNA coding for the -fragment of Escherichiacoli ß-galactosidase, and identified 176 differentbase substitution mutations by sequencing. The present methodgives mutant yields of 40–60%, when the mutants containabout one amino acid change per protein molecule. All typesof base substitution mutations can be generated and deletionsare rare. The efficiency of this method permits the use of relativelyelaborate screening systems to isolate mutants of either structuralgenes or regulatory regions.     

Biosynthesis of winter flounder antifreeze proprotein in E.coli

A semisynthetic winter flounder antifreeze proprotein (proAFP)coding region was constructed and inserted into a lacZ expressionvector. ProAFP was produced from the vector in Escherichia colias a C-terminal fusion to the first 289 amino acids of ß-galactosidase(ß-gal). The proAFP and ß-gal domains ofthe ß-gal–proAFP fusion protein were separatedby the recognition signal for the blood coagulation protease,factor X_a. Upon induction with isopropylthio-ß-D-galactosidethe fusion protein accumulated to levels of 15% of the totalprotein. The ß-gal–proAFP fusion protein waspartially purified by differential centrifugation, but requiredsolubilization prior to factor X_a digestion. The solubilizedfusion protein was efficiently and correctly cleaved by factorX_a, after which the proAFP was purified by gel permeation. BacterialproAFP was indistinguishable from natural proAFP by the criteriaof antifreeze activity, amino-terminal sequence (15 cycles),reverse-phase HPLC and SDS–polyacrylamide gel electrophoresis.Circular dichroism measurements showed that proAFP is a compositeof random coil and -helical secondary structure, with an -helicalcontent of 44% at 0°C. It seems probable that the C-terminalregion of proAFP, which corresponds to the mature AFP protein,is mainly -helical, and that the N-terminal pro-segment is randomcoiled.     

Spectroscopic investigation of structure in octarellin (a de novo protein designed to adopt the {alpha}/{beta}-barred packing)

We present here a spectroscopic structural characterizationof octarellin, a recently reported de novo protein modelledon /ß-barrel proteins [K. Go raj, A.Renard and J.A.Martial(1990) Protein Engng, 3, 259–266]. Infrared and Ramanspectra analyses of octarellin‘s secondary structure revealthe expected percentage of -helices (30%) and a higher ß-sheetcontent (40%) than predicted from the design. When the Ramanspectra obtained with octarellin and native triosephosphateisomerase (a natural /ß-barrel) are compared, similarpercentages of secondary structures are found. Thermal denaturationof octarellin monitored by CD confirms that its secondary structuresare quite stable, whereas its native-like tertiary fold is not.Tyrosine residues, predicted to be partially hidden from solvent,are actually exposed as revealed by Raman and UV absorptionspectra. We conclude that the attempted /ß-barrelconformation in octarellin may be loosely packed. The criteriaused to design octarellin are discussed and improvements suggested.     

Functional roles and subsite locations of Leu177, Trp178 and Asn182 of Aspergillus awamori glucoamylase determined by site-directed mutagenesis

Fungal glucoamylases contain four conserved regions. One regionfrom the Aspergillus niger enzyme contains three key carboxylicacid residues, the general acid catalytic group, Glu179, alongwith Asp176 and Glu180. Three site-directed mutations, Leu177– His, Trp178 – Arg and Asn182 – Ala, wereconstructed near these acidic groups to reveal the functionof other conserved residues in this region. Leu177 and Trp178are strictly conserved among fungal glucoamylases, while anamide, predominantly Asn, always occurs at position 182. Substitutionsof Leu177 or Trp178 cause significant decreases in k_cat withthe substrates tested. Similar increases in activation energiesobtained with Leu177 – His with both -(1,4)- and -(1,6)-linkedsubstrates indicate Leu177 is located in subsite 1. K_M valuesobtained with the Trp178 – Arg mutation increase for an-(1,6)-linked substrate, but not for -(1,4)-linked substrates.Calculated differences in activation energy between substratesindicate Trp178 interacts specifically with subsite 2. The Asn182 Ala mutation did not change k_cat or K_M values, indicating thatAsn182 is not crucial for activity. These results support amechanism for glucoamylase catalytic activity consisting ofa fast substrate binding step followed by a conformational changeat subsite 1 to stabilize the transition state complex.     

Cooperative deformation of a de novo designed protein

A de novo protein design has been made to understand the uniquepacking of natural proteins that have a ß/-barrelfold. A carefully designed 207 amino acid sequence was synthesizedusing an Escherichia coli expression system and the structuraland thermodynamic characteristics of the purified protein werestudied. At neutral pH the protein is soluble and monomeric,with large amounts of secondary structure and a hydrophobiccore, although the broad resonance peaks of its NMR spectrumsuggest that the designed protein does not have a unique structurewith tightly packed side chains. In an H–D exchange experiment,no amido protons of the designed protein exchanged slowly withdeuterons. At acidic pH, thermal unfolding was observedwitha remarkable change in the excess heat capacity measured directlyby a differential scanning microcalorimeter. The enthalpy andentropy differences at 110°C, extrapolated from analyzedthermodynamic parameters, are 1/3 of the common values for naturalproteins. These measurements indicate that the folding is significantlycooperative as expected, but that the protein is still looselypacked.     

A method for computational combinatorial peptide design of inhibitors of Ras protein

A computational combinatorial approach is proposed for the designof a peptide inhibitor of Ras protein. The procedure involvesthree steps. First, a `Multiple Copy Simultaneous Search' identifiesthe location of specific functional groups on the Ras surface.This search method allowed us to identify an important bindingsurface consisting of two ß strands (residues 5–8and 52–56), in addition to the well known Ras effectorloop and switch II region. The two ß strands had not previouslybeen reported to be involved in Ras–Raf interaction. Second,after constructing the peptide inhibitor chain based on thelocation of N-methylacetamide (NMA) minima, functional groupsare selected and connected to the main chain C atom. This stepgenerates a number of possible peptides with different sequenceson the Ras surface. Third, potential inhibitors are designedbased on a sequence alignment of the peptides generated in thesecond step. This computational approach reproduces the conservedpattern of hydrophobic, hydrophilic and charged amino acidsidentified from the Ras effectors. The advantages and limitationsof this approach are discussed.     

The structure of a designed peptidomimetic inhibitor complex of {alpha}-thrombin

Thrombin displays remarkable specificity, effecting the removalof fibrinopeptides A and B of fibrinogen through the selectivecleavage of two Arg–Gly bonds between the 181 Arg/Lys–Xaabonds in fibrinogen. Significant advances have been made inrecent years towards understanding the origin of the specificityof cleavage of the Argl6–Gly17 bond of the A-chain ofhuman fibrinogen. We have previously proposed a model for thebound structure of fibrinopeptide A_7–16 (FPA), based uponNMR data, computer-assisted molecular modeling and the synthesisand study of peptidomimetic substrates and inhibitors of thrombin.We now report the structure of the ternary complex of an FPAmimetic (FPAM), hirugen and thrombin at 2.5 Å resolution(R-factor = 0.138) and specificity data for the inhibition ofthrombin and related trypsin-like proteinases by FPAM. The crystallographicstructures of FPA and its chloromethyl ketone derivative boundto thrombin were determined. Although there are differencesbetween these structures in the above modeled FPA structureand that of the crystal structure of FPAM bound to thrombin,the , angles in the critical region of P1–P2–P3in all of the structures are similar to those of bovine pancreatictrypsin inhibitor (BPTI) in the BPTI–trypsin complex andD–Phe–Pro–Arg (PPACK) in the PPACK–thrombinstructure. A comparison between these and an NMR-derived structureis carried out and discussed.     

Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45

By chemoenzymatic synthesis the gene for a (Leu²⁷) analogueof human growth hormone releasing hormone-Gly⁴⁵ [(Leu²⁷GHRH-Gly⁴⁵]was constructed, cloned and expressed in Escherichia coli asa fusion protein with ß-galactosidase under the controlof the lac promoter and operator. Upon induction with isopropyl-D-thio-ß-galactopyranosidethe fusion protein accumulated to a yield of 15–20% ofthe total cellular protein. After cyanogen bromide deavage ofthe fusion protein the precursor peptide (Leu²⁷)hGHRH-Gly⁴⁵was separated by extraction and purified by ion exchange andh.p.l.c.-RP18 chromatography. The purified peptide was analysedby sequencing, isolectric focusing, amino acid analysis andamino acid analysis after V8 protease digestion. The carboxy-terminalglydne was subsequently amidated by PAM (peptidylglycine--amidating-monooxygenase),an enzyme which was isolated and characterized from fresh bovinepituitaries. Correct amidatlon of the penultimate amino acid,leucine, was verified by peptide sequencing with an authenticleucine amide reference.     

Structure-function relationships in the catalytic and starch binding domains of glucoamylase

Sixteen primary sequences from five sub-families of fungal,yeast and bacterial glucoamylases were related to structuralinformation from the model of the catalytic domain of Aspergillusawamori var. X100 glucoamylase obtained by protein crystallography.This domain is composed of thirteen -belices, with five conservedregions defining the active site. Interactions between methyl-maltoside and active site residues were modelled, and the importanceof these residues on the catalytic action of different glucoamylaseswas shown by their presence in each primary sequence. The overallstructure of the starch binding domain of some fungal glucoamylaseswas determined based on homology to the Cterminal domains ofBacillus cyclodextrin glucosyltransferases. Crystallographyindicated that this domain contains 6–8 ß-strandsand homology allowed the attribution of a disulfide bridge inthe glucoamylase starch binding domain. Glucoamylase residuesThr525, Asn530 and Trp560, homologous to Bacillus stearothermophiluscyclodextrin glucosyltransferase residues binding to maltosein the Cterminal domain, could be involved in raw-starch binding.The structure and length of the linker region between the catalyticand starch binding domains in fungal glucoamylases can varysubstantially, a further indication of the functional independenceof the two domains.     

Molecular modeling of the amyloid-{beta}-peptide using the homology to a fragment of triosephosphate isomerase that forms amyloid in vitro

The main component of the amyloid senile plaques found in Alzheimer'sbrain is the amyloid-ß-peptide (Aß), a proteolyticproduct of a membrane precursor protein. Previous structuralstudies have found different conformations for the Aßpeptide depending on the solvent and pH used. In general, theyhave suggested an -helix conformation at the N-terminal domainand a ß-sheet conformation for the C-terminal domain.The structure of the complete Aß peptide (residues 1–40)solved by NMR has revealed that only helical structure is presentin Aß. However, this result cannot explain the large ß-sheetAß aggregates known to form amyloid under physiologicalconditions. Therefore, we investigated the structure of Aßby molecular modeling based on extensive homology using theSmith and Waterman algorithm implemented in the MPsrch program(Blitz server). The results showed a mean value of 23% identitywith selected sequences. Since these values do not allow a clearhomology to be established with a reference structure in orderto perform molecular modeling studies, we searched for detailedhomology. A 28% identity with an /ß segment of a triosephosphateisomerase (TIM) from Culex tarralis with an unsolved three-dimensionalstructure was obtained. Then, multiple sequence alignment wasperformed considering Aß, TIM from C.tarralis and anotherfive TIM sequences with known three-dimensional structures.We found a TIM segment with secondary structure elements inagreement with previous experimental data for Aß. Moreover,when a synthetic peptide from this TIM segment was studied invitro, it was able to aggregate and to form amyloid fibrils,as established by Congo red binding and electron microscopy.The Aß model obtained was optimized by molecular dynamicsconsidering ionizable side chains in order to simulate Aßin a neutral pH environment. We report here the structural implicationsof this study.     

Strong inhibition of fibrinogen binding to platelet receptor {alpha}IIb{beta}3 by RGD sequences installed into a presentation scaffold

In order to probe the structural constraints on binding of RGDsequences to the platelet receptor _IIbß₃ we have usedrecombinant DNA techniques to install the RGD sequence into‘presentation scaffolds’, small proteins of known3-D structure chosen to present guest sequences in constrainedorientations. Using Escherichia coli expression systems we madesequence variants in which loop residues of the immunoglobulinV_L domain REI and of human interleukin-1ß were replaced(without changing polypeptide length) by the RGD sequence atpositions predicted, based on small molecule studies, to orientthe RGD moiety into an active conformation. These variants donot compete for fibrinogen binding to _IIbß₃ up toalmost 1 mM concentration. Unfolded or proteolytically fragmentedforms of these same proteins do compete, however, showing thatthe RGD sequences in the mutants must be prohibited from bindingby constraints imposed by scaffold structure. To suppress theeffects of such structural constraints we constructed two sequencevariants in which RGD-containing sequences 42–57 or 44–55from the snake venom platelet antagonist kistrin were inserted(this increasing the length of the loop) into the third complementaritydetermining loop of REI. Both of these variants compete stronglyfor fibrinogen binding with IC₅₀s in the nM range. These results,plus data on kistrin-related peptides also presented here, suggestthat the molecular scaffold REI is capable of providing to aninstalled sequence a structural context and conformation beneficialto binding. The results also suggest that in order to bind wellto _IIbß₃, RGD sequences in protein ligands must eitherproject significantly from the surface of the scaffold and/orretain a degree of conformational flexibility within the scaffold.Molecular scaffolds like REI should prove useful in the elucidationof structure-function relationships and the discovery of newactive sequences, and may also serve as the basis for noveltherapeutic agents.     

Molecular modeling of coiled-coil {alpha}-tropomyosin: analysis of staggered and in register helix--helix interactions

In register and staggered models of tropomyosin coiled-coilwere built from X-ray C coordinates and refined via moleculardynamics. The two models show similar structural features withthe X-ray structure of GCN4 leucine zipper. Empirical energeticmethods used to compare the in register and staggered modelsindicate that both are equally probable. The two models havesimilar profiles of solvation free energy of folding for residuesat positions a and d of the repeating heptad, indicating thatresidues at these positions are as well buried in an in registerstructure as in a staggered one. Neither the in register northe 14 residues staggered structure can be ruled out based onhydrophobic or e–g' (g–e') electrostatic interactionswhich are not able to distinguish between the two models andare therefore not selective. However, the eg–b'c' electrostaticinteractions, although smaller in magnitude, are in favor ofthe in register model. Furthermore, analysis of hydrophobicand electrostatic interactions along the tropomyosin sequenceshows that bulky residues in positions a and d prevent the formationof inter-chain salt bridges.