Hydrophobic effects on protein/nucleic acid interaction: enhancement of substrate binding by mutating tyrosine 45 to tryptophan in ribonuclease Tl

Hydrophobic effects on binding of ribonuclease Tl to guaninebases of several ribonucleotides have been proved by mutatinga hydrophobic residue at the recognition site and by measuringthe effect on binding. Mutation of a hydrophobic surface residueto a more hydrophobic residue (Tyr45 – Trp) enhances thebinding to ribonucleotides, including mononucleotide inhibitorand product, and a synthetic substrate-analog trinudeotide aswell as the binding to dinucleotide substrates and RNA. Enhancementson binding to non-substrate ribonucleotides by the mutationhave been observed with free energy changes ranging from –2.2 to – 3 .9 kJ/mol. These changes are in good agreementwith that of substrate binding, –2.3 kJ/mol, which iscalculated from Michaelis constants obtained from kinetic studies.It is shown, by comparing the observed and calculated changesin binding free energy with differences in the observed transferfree energy changes of the amino acid side chains from organicsolvents to water, that the enhancement observed on guaninebinding comes from the difference in the hydrophobic effectsof the side chains of tyrosine and tryptophan. Furthermore,a linear relationship between nucleolytic activities and hydrophobicityof the residues (Ala, Phe, Tyr, Trp) at position 45 is observed.The mutation could not change substantially the base specificityof RNase Tl, which exhibits a prime requirement for guaninebases of substrates.     

Hydrophobic regions on protein surfaces: definition based on hydration shell structure and a quick method for their computation

The hydrophobic part of the solvent-accessible surface of atypical monomeric globular protein consists of a single, largeinterconnected region formed from faces of apolar atoms andconstituting –60% of the solvent-accessible surface area.Therefore, the direct delineation of the hydrophobic surfacepatches on an atom-wise basis is impossible. Experimental dataindicate that, in a two-state hydration model, a protein canbe considered to be unified with its first hydration shell inits interaction with bulk water. We show that, if the surfacearea occupied by water molecules bound at polar protein atomsas generated by AUTOSOL is removed, only about two-thirds ofthe hydrophobic part of the protein surface remains accessibleto bulk solvent. Moreover, the organization of the hydrophobicpart of the solvent-accessible surface experiences a drasticchange, such that the single interconnected hydrophobic regiondisintegrates into many smaller patches, i.e. the physical definitionof a hydrophobic surface region as unoccupied by first hydrationshell water molecules can distinguish between hydrophobic surfaceclusters and small interconnecting channels. It is these remaininghydrophobic surface pieces that probably play an important rolein intraand intermolecular recognition processes such as ligandbinding, protein folding and protein–protein associationin solution conditions. These observations have led to the developmentof an accurate and quick analytical technique for the automaticdetermination of hydrophobic surface patches of proteins. Thistechnique is not aggravated by the limiting assumptions of themethods for generating explicit water hydration positions. Formationof the hydrophobic surface regions owing to the structure ofthe first hydration shell can be computationally simulated bya small radial increment in solvent-accessible polar atoms,followed by calculation of the remaining exposed hydrophobicpatches. We demonstrate that a radial increase of 0.35–0.50Å resembles the effect of tightly bound water on the organizationof the hydrophobic part of the solvent-accessible surface.     

Extracting hydrophobicity parameters from solute partition and protein mutation/unfolding experiments

Hydrophobicity values for amino acids obtained from proteinunfolding experiments are about twice as large as those obtainedfrom data on the partitioning of amino acids between water andoctanol. Quantitative analyses of several data sets, presentedhere, indicate that the difference is best explained by themost direct hypothesis, i.e. that the environment of hydrophobicgroups in the interior of a protein is poorly modeled by octanol.Instead, we propose—and provide supporting evidence—thathydrocarbons are a more suitable model. First, we reanalyzedata from both solute partitioning and protein unfolding experiments,taking account of the effects that were omitted previously,by introducing a volume dependence in the former and a fullfree energy analysis in the latter. Both changes in evaluationmethodology decrease the discrepancy, but the differences remainsubstantial. The hydrophobicity parameter obtained from side–chaintransfers between octanol and water increases from 16.7 to 22cal/ mol/², while that obtained from protein unfolding decreasesfrom 34.9 to 31.2 cal/mol/². On the other hand,our analysisof the solubilities of pure hydrocarbons in water provides ahydrophobicity parameter of 30.8 cal/mol/². This apparent hydrocarbon–likeenvironment of a protein's interior is also suggested more directlyby an analysis of the contact environment of hydrophobic sidechains in mutation/unfolding experiments, which have polar contactareas that are     

Contribution of a disulfide bridge to the stability of 1,3-1,4-P-D-glucan 4-glucanohydrolase from Bacillus licheniformis

Bacillus 1,3-1,4-ß-glucanases possess a highly conserveddisulfide bridge connecting a ß-strand with a solventexposedloop lying on top of the extended binding site cleft The contributionof the disulfide bond and of both individual cysteines (Cys61and Cys90) in the Bacillus licheniformis enzyme to stabilityand activity has been evaluated by protein engineering methods.Reduction of the disulfide bond has no effect on kinetic parameters,has only a minor effect on the activity-temperature profileat high temperatures, and destabilizes the protein by less than0.7 kcal/mol as measured by equilibrium urea denatu ration at37°C. Replacing either of the Cys residues with Ala destabilizesthe protein and lowers the specific activity. C90A retains 70%of wild-type (wt) activity (in terms of Vmax), whereas C61Aand the double mutant C61A–C90A have 10% of wt V_max. Alarger change in free energy of unfolding is seen by equilibriumurea denaturation for the C61A mutation (loop residue, 3.2 kcal/molrelative to reduced wt) as compared with the C90A mutation (ß-strandresidue, 1.8 kcal/mol relative to reduced wt), while the doublemutant C61A–C90A is 0.8 kcal/mol less stable than thesingle C61A mutant. The effects on stability are interpretedas a result of the change in hydrophobic packing that occursupon removal of the sulfur atoms in the Cys to Ala mutations     

Interchain cysteine bridges control entry of progesterone to the central cavity of the uteroglobin dimer

The progesterone–binding protein uteroglobin has beenexpressed in Escherichia coli in an unfused, soluble form. likemature uteroglobin from rabbit endometrium (UG), the E.coliproduceduteroglobin (UG1) dimerizes in vitro, forms an antiparalleldimer with Cys3–Cys69' and Cys69–Cys3' disulfidebonds and binds progesterone under reducing conditions. In orderto analyze the dimerization and the reduction dependence ofprogesterone binding in more detail, we separately replacedcysteine 3 and cysteine 69 by serines. Under reducing conditions,both uteroglobin variants (UGl–3Ser and UGl–69Ser)bind progesterone with the same affinity as the wild–typesuggesting that both cysteine residues are not directly involvedin progesterone binding. In contrast to the wild–typeprotein, both cysteine variants also bind progesterone withhigh affinity in the absence of reducing agents. In addition,UGl-3Ser and UGl-69Ser both form covalently linked homodimers.Thus, unnatural Cys69–69' and Cys3–3' disulfidebonds exist in UG1–3Ser and UG1–69Ser, respectively.These data together with computer models based on X-ray diffractiondata strongly support the idea that progesterone reaches itsbinding site located in an internal hydrophobic cavity via ahydrophobic tunnel along helices 1 and 4. Under non–reducingconditions the tunnel is closed by two disulfide bridges (Cys3–Cys69'(and Cys69–Cys3') that lie in the most flexible regionof the dimer. Reduction or replacement of a cysteine residueenables conformational changes that open the channel allowingprogesterone to enter.     

Molecular mechanics analysis of inhibitor binding to HIV-1 protease

Crystallographic structures of HIV protease with three differentpeptide-mimetic inhibitors were subjected to energy minimizationusing molecular mechanics, the minimized structures analyzedand the inhibitor binding energies calculated. Partial chargeassignment for the hydrogen bonded catalytic aspartk acids,Asp25 and -25', was in good agreement with charge calculationsusing semi-empirical molecular orbital methods. Root mean squaredeviations on minimization were small and similar for both subunitsin the protease dimer. The surface loops, which had the largestB factors, changed most on minimization; the hydrophobic coreand the inhibitor binding site showed little change. The distance-dependentdielectric of D(r) = 4r was found to be preferable to D(r) =r. Distance restraints were applied for the intermolecular hydrogenbonds to maintain the conformation of the inhibitor bindingsite. Using the dielectric of D(r) = 4r, the calculated interactionenergy of the three inhibitors with the protease ranged from–53 to –56 kcal/mol. The groups of the inhibitorswere changed to add or remove a ‘transition state analogue’hydroxyl group, and the loss in energy on the removal of thisgroup was calculated to be 0.9–1.7 kcal/mol. This wouldrepresent 19–36% of the total measured difference in bindingenergy between the inhibitors JG365 and MVT-101.     

Molecular modelling and site-directed mutagenesis on a bovine anti-testosterone monoclonal antibody

A three-dimensional (3D) molecular model of the antigen-combiningsite of a bovine anti-testosterone monoclonal antibody has beenconstructed. In the model, the CDRs, and a single heavy chainframework region residue (Trp47), associate to form a hydrophobiccavity large enough to accommodate a single molecule of testosterone.Tyr97 of CDR-H3 lies at the bottom of the cavity with its hydroxylgroup exposed to solvent. Using the model and data from bindingstudies, we predicted that the cavity forms the antibody's paratopeand on binding testosterone a hydrogen bond is formed betweenTyr97 of CDR-H3 and the hydroxyl group on the D-ring of testosterone.This prediction has subsequently been tested by site-directedmutagenesis. An antibody with phenylalanine in place of tyrosineat position 97 in CDR-H3 has its affinity reduced by {smalltilde}800 fold. The reduction in binding energy associated withthe reduced affinity has been calculated to be 3.9 kcal/molwhich is within the range (0.5–4.0 kcal/mol) expectedfor the loss of a single hydrogen bond. The model has been usedto suggest ways of increasing the antibody's affinity for testosterone.     

The effect of engineering surface loops on the thermal stability of Bacillus subtilis neutral protease

Using genetic techniques the contribution of surface loops tothe thermal stability of Bacillus subtUis neutral protease (NPsub)wasstudied. Mutations were designed to make the surface of NP-submore similar to the surface of more thermostable neutral proteasessuch as thermolysin (TLN). The mutations included the replacementof an irregular loop by a shorter variant and the introductionof a ten–residue (3– hairpin. In general, thesedrastic mutations had little effect on the production and activityof NP–sub, indicating the feasibility of major structuralrearrangements at the surface of proteins. In the most stablemutant, exhibiting an increase in thermal stability of 1.1°C, 10% of the surface of NP–sub was modified. Several NP–subvariants carrying multiple mutations were constructed. Non–additiveeffects on thermal stability were observed, which were interpretedon the basis of a model for thermal inactivation, that emphasizesthe importance of local unfolding processes for thermal stability     

Free energy simulations of the HyHEL-10/HEL antibody-antigen complex

Free energy simulations are reported for the N31^L-D mutation,both in the HyHEL-10-HEL antibody-lysozyme complex and in theunliganded antibody, using the thermo-dynamic-cycle perturbationmethod. The present study suggests that the mutation would changethe free energy of binding of the complex by –5.6 kcal/mol(unrestrained free energy simulations), by –0.5 kcal/mol(free energy simulations with a restrained backbone) and by1.8 kcal/ mol (Poisson-Boltzmann calculations, which also usea restrained geometry model). A detailed structural analysishelps in estimating the contributions from various residuesand regions of the system. Enhanced recognition of HEL by themutant HyHEL-10 would arise from the combination of thermodynamicallymore favorable conformational changes of the CDR loops uponassociation and subsequent charge pairing with Lys96 in theantigen.     

Stabilization of lysozyme against irreversible inactivation by alterations of the Asp-Gly sequences

Site-directed mutagenesis was performed at Asp-Gly (48–49,66–67, 101–102) and Asn-Gly (103–104) sequencesof hen egg-white lysozyme to protect the enzyme against irreversiblethermoinactivation. Because the lysozyme inactivation was causedby the accumulation of multiple chemical reactions, includingthe isomerization of the Asp-Gly sequence and the deamidationof Asn [Tomizawa et al.(1994) Biochemistry, 33, 13032–13037],the suppression of these reactions by the substitution of Glyto Ala, or the introduction of a sequence of human-type lysozyme,was attempted and the mutants (where each or all labile sequenceswere replaced) were prepared. The substitution resulted in thereversible destabilization from 1 to 2 kcal/mol per substitution.The destabilization was caused by the introduction of ß-carbonto the constrained position that had conformational angles withinthe allowed range for the Gly residue. Despite the decreasein the reversible conformational stability, the mutants hadmore resistance to irreversible inactivation at pH 4 and 100°C.In particular, the rate of irreversible inactivation of themutant, which was replaced at four chemically labile sequences,was the latest and corresponded to 18 kcal/mol of the reversibleconformational stability. Therefore, replacement of the chemicallylabile sequence was found to be more effective at protectingenzymes against irreversible thermoinactivation than at strengtheningreversible conformational stability.     

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.     

Predicting leucine zipper structures from sequence

The leucine zipper structure is adopted by one family of thecoiled coil proteins. Leucine zippers have a characteristicleucine repeat: Leu–X₆–Leu–X₆–Leu–X₆–Leu(where X may be any residue). However, many sequences have theleucine repeat, but do not adopt the leucine zipper structure(we shall refer to these as non-zippers). We have found andanalyzed residue pair patterns that allow one to identify correctly90% of leucine zippers and 97% of non-zippers. Simpler analyses,based on the frequency of occurrence of residues at certainpositions, specify, at most, 65% of zippers and 80–90%of non-zippers. Both short and long patterns contribute to thesuccessful discrimination of leucine zippers from non-zippers.A number of these patterns involve hydrophobic residues thatwould be placed on the solvent-exposed surface of the helix,were the sequence to adopt a leucine zipper structure. Thus,an analysis of protein sequences has allowed us to improve discriminationbetween leucine zippers and non-zippers, and has provided somefurther insight into the physical factors influencing the leucinezipper structure.     

The net energetic contribution of interhelical electrostatic attractions to coiled-coil stability

The net energetic contribution of interhelical electrostaticattractions to coiled-coil stability has been quantitated usingde novo designed synthetic coiled-coils. The synthesized modelcoiled-coil (EK), denoted by amino acid res-idues in positionse and g, which contains only interhelical ionic interactionswithout any possible (i, i + 3) and (i, i + 4) intrahelicalionic interaction, consists of two identical 35 residue polypeptidechains with a heptad repeat KgLaG-bAcLdEeKf. Three mutant coiled-coilswere prepared where five Glu residues at e positions in EK weremutated to Gin residues (QK); five Lys residues at g positionswere altered to Gin residues (EQ) or these mutations were effectedat both positions e and g (QQ). The stabilities of the fourcoiled-coils were determined by measuring the ellipticitiesat 220 nm as a function of urea concentration at 20C. By usinga double-mutant cycle analysis it was possible to isolate theenergetic contribution of interhelical ionic attractions tocoiled-coil stability from the other contributions such as helicalpreference and hydro-phobicity. The 0.37 0.01 kcal/mol ofenergetic contribution of one interhelical ion pair to the coiled-coilstability was obtained from three independent comparisons. Thisfinding suggests that a large number of weak interhelical electrostaticinteractions on the surface of a protein can make a substantialcontribution to protein stability. In addition, the energeticcontributions of a single mutation E^– Q, K⁺Q, Q E andE^– Ewere also determined (G = 0.22, 0.26, 0.46 and 0.65kcal/mol for the single mutations, respectively). The greatercontribution of a protonated Glu residue to coiled-coil stabilitycompared with an ionized Glu residue (0.65 kcal/mol) can outweighthe relatively smaller contribution of an interhelical ion pair(0.37 kcal/mol), which clearly explains why most coiled-coilsare more stable at acidic pH compared with neutral pH even wheninterhelical salt bridges contribute to the coiled-coil stabilityat neutral pH.     

Conformation equilibria of valine studies by dynamics simulation

The conformational probability distribution of a valine residuein the valine dipeptide and of the valine side chain in an -helix,as well as the change in helix stability for replacing alaninewith valine, has been calculated by molecular dynamics simulationsof explicitly hydrated systems: dipeptide, tetrapeptide and10-, 14- and 18-residue oligoalanine helices. All computed free-energydifferences are means from at least eight separate slow-growthsimulations, four in each direction and are reported with theirroot-meansquare deviations. Different values for the changein free energy of folding (G°) have been calculated withthe use of forcefields having an all-atom and a central-atomrepresentation of methyl groups, etc. The value obtained withthe all-atom forcefield agrees well with new experimental values(3 kJ/mol = 0.7 kcal/mol). Furthermore, the most stable valineside-chain rotamer in the helix is different for these two representations.The most stable rotamer for the all atom conformation is thesame one that predominates for valines in a-helices in proteinsof known conformation. The lower conformational freedom of thevaline side chain in the helix contributes 1 kJ/mol to the differencein stability computed with the all-atom potential; unfavorableinteractions of the side chain with the helix, even in the moststable conformation, further increase G°.     

Specificity determinants of rat tissue kallikrein probed by site-directed mutagenesis

Site-specific mutagenesis was employed to study structure-functionrelationships at the substrate binding site of rat tissue kallikrein.Four kallikrein mutants, the Pro219 deletion (P219del), the34–38 loop Tyr-Tyr-Phe-Gly to Ile-Asn mutation [YYFG(34–38)IN],the Trp215Gly exchange (W215G) and the double mutant with Tyr99Hisand Trp215Gly exchange (Y99H:W215G) were created by site-directedmutagenesis to probe their function in substrate binding. Themutant proteins were expressed in Esclzerichia coli at highlevels and analyzed by Western blot. These mutant enzymes werepurified to apparent homogeneity. Each migrated as a singleband on SDS-PAGE, with slightly lower molecular mass (36 kDa)than that of the native enzyme, (38 kDa) because of their lackof glycosylation. The recombinant kallikreins are immunologicallyidentical to the native enzyme, displaying parallelism withthe native enzyme in a direct radioimmunoassay for rat tissuekallikrein. Kinetic analyses of K_m and k_cat using fluorogenicpeptide substrates support the hypothesis that the Tyr99–Trp215interaction is a major determinant for hydrophobic P2 specificity.The results suggest an important role for the 34–38 loopin hydrophobic P3 affinity and further show that Pro219 is essentialto substrate binding and efficient catalysis of tissue kallikrein.     

Display of {beta}-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp'-OmpA'-{beta}-lactamase fusions

Bacterial cell-surface exposure of foreign peptides and solubleproteins has been achieved recently by employing a fusion proteinmethodology. An Lpp'–OmpA(46–159)–Bla fusionprotein has been shown previously to display the normally periplasmicenzyme ß-lactamase (Bla) on the cell surface of theGram-negative bacterium Escherichia coli. Here, we have investigatedthe role of the OmpA domain of the tripartite fusion proteinin the surface display of the passenger domain (Bla) and havecharacterized the effects of the fusion proteins on the integrityand permeability of the outer membrane. We show that in additionto OmpA(46–159), a second OmpA segment, consisting ofamino acids 46–66, can also mediate the display of Blaon the cell surface. Other OmpA domains of various lengths (aminoacids 46–84, 46–109, 46–128, 46–141and 46–145) either anchored the Bla domain on the periplasmicface of the outer membrane or caused a major disruption of theouter membrane, allowing the penetration of antibodies intothe cell. Detergent and antibiotic sensitivity and periplasmicleakage assays showed that changes in the permeability of theouter membrane are an unavoidable consequence of displayinga large periplasmic protein on the surface of E.coli. This isthe first systematic report on the effects that cell surfaceengineering may have on the integrity and permeability propertiesof bacterial outer membranes.     

Molecular modeling and mechanism of action of human decay-accelerating factor

A model of the regulatory region of human decay acceleratingfactor (DAF) was built based on the known coordinates of a fragmentof the structurally and functionally homologous serum protein,factor H. According to this model, the four short consensusrepeats (SCRs) in DAF are arranged in a helical fashion. A positivelycharged surface area on SCRs 2 and 3, two of the three repeatingunits essential for function, is postulated to be the primaryrecognition site for the C3 convertases C4b2a and C3bBb. Thisarea encompasses a cavity on SCR 2, as well as part of the grooveon the SCR 2–SCR 3 interface. Two additional surface depressionsare centered around the C-terminal disulfide bridges of SCRs3 and 4. These are likely to provide additional ligand bindingsites. Based on this model in conjunction with sequence homologyto the Ba fragment of factor B, a mechanism of DAF's acceleratedconvertase decay action is postulated.     

Comparison of conservation within and between the Ser/Thr and Tyr protein kinase family: proposed model for the catalytic domain of the epidermal growth factor receptor

The protein kinase family can be subdivided into two main groupsbased on their ability to phosphorylate Ser/Thr or Tyr substrates.In order to understand the basis of this functional difference,we have carried out a comparative analysis of sequence conservationwithin and between the Ser/Thr and Tyr protein kinases. A multiplesequence alignment of 86 protein kinase sequences was generated.For each position in the alignment we have computed the conservationof residue type in the Ser/Thr, in the Tyr and in both of thekinase subfamilies. To understand the structural and/or functionalbasis for the conservation, we have mapped these conservationproperties onto the backbone of the recently determined structureof the cAMP–dependent Ser/Thr kinase. The results showthat the kinase structure can be roughly segregated, based uponconservation, into three zones. The inner zone contains residueshighly conserved in all the kinase family and describes thehydrophobic core of the enzyme together with residues essentialfor substrate and ATP binding and catalysis. The outer zonecontains residues highly variable in all kinases and representsthe solvent–exposed surface of the protein. The thirdzone is comprised of residues conserved in either the Ser/Thror Tyr kinases or in both, but which are not conserved betweenthem. These are sandwiched between the hydrophobic core andthe solvent-exposed surface. In addition to analyzing overallconservation hi the kinase family, we have also looked at conservationof its substrate and ATP binding sites. The ATP site is highlyconserved throughout the kinases, whereas the substrate bindingsite is more variable. The active site contains several positionswhich differ between the Ser/Thr and Tyr kinases and may beresponsible for discriminating between hydroxyl bearing sidechains. Using this information we propose a model for Tyr substratebinding to the catalytic domain of the epidermal growth factorreceptor (EGFR).     

Stability of native and covalently modified papain

Covalent modification of enzymes with large polymers can producemodified enzymes which retain considerable biological activityand at the same time display resistance to denaturation by hightemperatures and chaotropic agents. The cysteine protease, papain,with potential applications in industry, was covalently coupledto polymeric sucrose (mol. wt 400 kDa) at different ratios.The derivatives retained >80% intrinsic catalytic activitywith no change in pH optima and kinetic constants, indicatingthat the gross tertiary structure was not altered by modification.However, they displayed better thermotolerance than native papain,as indicated by their higher T₅₀ values (6–10°C) andtheir temperature optima being shifted by 10°C. The half-lifeof modified papain, calculated from the rate of thermoinactivation,was prolonged by 2- to 30-fold over the native depending onthe temperature and proportion of polymeric sucrose in the adducts.The increases in activation free energy of inactivation (1–10kj/mol) and activation enthalpy (4-78 kj/mol) indicate stabilizationof the protein and lesser inactivation due to spontaneous unfolding.In the presence of urea, modified papain showed activation,which may be due to a loosening of the ‘rigid’ structure,reminiscent of the property of thermophilic enzymes.     

Correlation of surface properties with conformational stabilities of wild-type and six mutant tryptophan synthase {alpha}-subunits substituted at the same position

The surface properties of wild-type and six mutant -subunitsof tryptophan synthase substituted at the same position, 49,which is buried in the interior, were measured by surface tension,foaming and emulsifying properties to correlate the surfaceproperties with the stabilities. The conformational stabilitiesof the seven -subunits differed dramatically depending on thecharacteristics of the substituting residues [Yutani et al.(1987) Proc. Natl. Acad. Sci., 84, 4441–4444]. The mutantproteins substituted by isoleucine and phenylalanine in placeof glutamic acid at position 49 were more stable than the otherproteins and showed higher surface tension and lower foamingand emulsifying properties than the wild-type and other mutantproteins. Good correlations were observed between these surfaceproperties and values of the Gibbs free energy of unfoldingin water, of the proteins. This indicates that the surface propertiesof the -subunits of tryptophan synthase depend closely on theconformational stabilities.