Experimental verification of the `stability profile of mutant protein' (SPMP) data using mutant human lysozymes

The stability profile of mutant protein (SPMP) (Ota,M., Kanaya,S.and Nishikawa,K., 1995, J. Mol. Biol., 248, 733–738) estimatesthe changes in conformational stability due to single aminoacid substitutions using a pseudo-energy potential developedfor evaluating structure–sequence compatibility in thestructure prediction method, the 3D–1D compatibility evaluation.Nine mutant human lysozymes expected to significantly increasein stability from SPMP were constructed, in order to experimentallyverify the reliability of SPMP. The thermodynamic parametersfor denaturation and crystal structures of these mutant proteinswere determined. One mutant protein was stabilized as expected,compared with the wild-type protein. However, the others werenot stabilized even though the structural changes were subtle,indicating that SPMP overestimates the increase in stabilityor underestimates negative effects due to substitution. Thestability changes in the other mutant human lysozymes previouslyreported were also analyzed by SPMP. The correlation of thestability changes between the experiment and prediction dependedon the types of substitution: there were some correlations forproline mutants and cavity-creating mutants, but no correlationfor mutants related to side-chain hydrogen bonds. The presentresults may indicate some additional factors that should beconsidered in the calculation of SPMP, suggesting that SPMPcan be refined further.     

Selection of a thermostable variant of chloramphenicol acetyltransferase (Cat-86)

The moderate thermophile Bacillus stearothermophilus was usedas a host in which to detect more thermostable variants of theB.pumilus chloramphenicol acetyltransferase (Cat-86) protein.Seventeen mutants were isolated and detected by their abilityto grow in the presence of chloramphenicol at a previously restrictivetemperature (58°C). The genes encoding these proteins weresequenced; all 17 mutants carried the same C to T transitionthat conferred an amino acid substitution of alanine by valineat position 203 of the protein sequence. The wild-type and onemutant Cat-86 protein were purified to homogeneity using affinitychromatography, and kinetic and thermal stability studies wereundertaken. Both enzymes had similar sp. act. in the regionof 215 U/mg, with K_m values for chloramphenicol in the range13.8–15.4 µM and for acetyl CoA in the range 13.6–15.5µM. The A203V mutant shows greater stability than thewild-type Cat-86 protein at temperatures above 50°C andappears to pass through a transition state between 48 and 50°C.     

Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl--disulfide interchange

In the preceding paper in this issue, we described the overproduction of one mutant chicken lysozyme in Escherichia coil.Since this lysozyme contained two amino acid substitutions (Ala³¹ValandAsn¹⁰⁶Ser)in addition to an extra methionine residue at theNH₂-terminus the substituted amino acid residues were convertedback to the original ones by means of oligonucleotide-directedsite-specific mutagenesis and in vitro recombination. Thus fourkinds of chicken lysozyme [Met^–1 Val³¹Ser¹⁰⁶-, Met^–1Ser¹⁰⁶-,Met^–1 Val³¹-and Met^–1 (wild type)] wereexpressed in E. coli. From the results of folding experimentsof the reduced lysozymes by sulfhydryl-disulfide interchangeat pH 8.0 and 38°C, follow ed by the specific activity measurementsof the folded en zymes, the following conclusions can be drawn:(i) an extra methionine residue at the NH₂-terminus reducesthe folding rate but does not affect the lysozyme activity ofthe folded enzyme; (ii) the substitution of Asn¹⁰⁶ by Ser decreasesthe activity to 58% of that of intact native lysozyme withoutchanging the folding rate; and (iii) the substitution of Ala³¹Val prohibits the correct folding of lysozyme. Since the wildtype enzyme (Met^–1-lysozyme) was activated in vitro withoutloss of specific activity, the systems described in this study(mutagenesis, overproduction, purification and folding of inactivemutant lysozymes) may be useful in the study of folding pathways,expression of biological activity and stability of lysozyme.     

Cumulative stabilizing effects of hydrophobic interactions on the surface of the neutral protease from Bacillus subtilis

Using genetically engineered mutants of the neutral pro-teasefrom Bacillus stearothermophilus (BsteNP), it had been shownthat the surface-exposed structural motif constituted by Phe63embedded in a four amino acid hydrophobic pocket is criticalfor the thermal stability of the thermophilic neutral proteasesfrom Bacilli. To measure the stabilizing contribution of eachhydrophobic interaction taking place between Phe63 and the hydrophobicpocket, we grafted this structural motif in the neutral proteasefrom the mesophile Bacillus subtilis (BsubNP). This was accomplishedby first creating the Thr63Phe mutant of BsubNP and then generatinga series of mutants in which the four amino acids which in thermolysinsurround Phe63 and form the hydrophobic pocket were added oneafter the other. By analysing the thermal stability of eachmutant it was found that the 2°C destabilizing effect ofthe Thr63Phe substitution was completely suppressed by the additionof the four amino acid hydrophobic pocket, each replacementproviding a stabilizing contribution of approxi mately 0.8–1°C.These results are discussed in the light of the peculiar mechanismof thermal inactivation of proteolytic enzymes.     

The active site of carboxypeptidase Taq possesses the active-site motif His-Glu-X-X-His of zinc-dependent endopeptidases and aminopeptidases

Carboxypeptidase (CPase) Taq possesses the His–Glu–X–X–Hissequence, which is the consensus sequence in the active siteof zinc-dependent endopeptidases and amino-peptidases, at positions276–280. Amino acid replacement of the conserved His andGlu drastically diminished the activity of CPase Taq, and thezinc content of the enzyme was also greatly reduced when eitherof the two His residues was replaced with Arg or Tyr. The resultsindicate that this sequence actually functions as the activesite in CPase Taq, showing that CPase Taq is a novel type ofzinc-dependent CPase that possesses the His–Glu–X–X–Hisactive-site motif.     

Stabilization of lysozyme by the introduction of Gly-Pro sequence

Three mutant lysozymes where the Asp¹⁰¹ – Gly¹⁰² sequenceof lysozyme was converted to Asp¹⁰¹–Pro¹⁰², Gly¹⁰¹–Pro102and Pro¹⁰¹–Gly¹⁰² were prepared to investigate the effectof proline residues on the stabilization of proteins. The freeenergy changes of lysozymes for the unfolding in aqueous solutionat pH 5.5 and 35°C were 10.0, 10.1, 11.0 and 7.7 kcal/molfor wild type, Asp¹⁰¹Pro¹⁰², Gly¹⁰¹Pro¹⁰² and Pro¹⁰¹Gly¹⁰² lysozymerespectively. When the energy level in the unfolded state ofwild type lysozyme was fixed at a standard level, the energylevels in the folded state of Asp¹⁰¹Pro¹⁰² and Pro¹⁰¹Gly¹⁰²lysozymes were found to be higher than that of wild type lysozymeon the basis of G_D(H₂O) and entropy losses of their polypeptidechains in the unfolded state. The presence of some strain inthe folded state of these lysozymes was supported by both thecalculation of conformational energy for a trans-L-prolyl residue[Schimmel, P.R. and Flory,P.J. (1968) J. Mol. Biol, 34, 105–120] and the analysis of structures of energy-minimizedmutant lysozymes. Therefore, it is concluded that the formationof the Gly-Pro sequence is effective in avoiding possible strainin the folded state of a protein caused by the introductionof proline residue(s).     

Effect of Lys->Arg mutation on the thermal stability of Cu,Zn superoxide dismutase: influence on the monomer-dimer equilibrium

The thermal stability of two single (K3R, K67R) and one double(K3R-K67R) mutants of Xenopus laevis B Cu,Zn superoxide dismutasehas been studied to test LysArg substitution as an ‘electrostaticallyconservative’ strategy to increase protein stability.The K3R mutant displays an increased thermostability with respectto the wild-type enzyme, whilst a decreased stability was observedin the case of the K67R and K3R-K67R mutants. Concentrationdependence of the apparent inactivation constant (k_app) of thelatter mutants, as compared to that of the wild type enzymeand K3R mutant, indicates that their higher sensitivity to heatinactivation is due to a perturbation of the dimer association.These results are confirmed also by fluorescence anisotropymeasurements of the internal probe Tyr149. The possible roleof Arg67 in perturbing the dimer dissociation equilibrium towardthe monomeric form is discussed.     

Analysis of a catalytic pathway via a covalent adduct of D52E hen egg white mutant lysozyme by further mutation

We previously demonstrated by X-ray crystallography and electrospraymass spectrometry that D52E mutant hen lysozyme formed a covalentenzyme–substrate adduct on reaction with N-acetylglucosamineoligomer. This observation indicates that D52E lysozyme mayacquire a catalytic pathway via a covalent adduct. To explainthis pathway, the formation and hydrolysis reactions of thecovalent adduct were investigated. Kinetic analysis indicatedthat the hydrolysis step was the rate-limiting step, 60-foldslower than the formation reaction. In the formation reaction,the pH dependence was bell-shaped, which was plausibly explainedby the functions of the two catalytic pK_as of Glu35 and Glu52.On the other hand, the pH dependence in the hydrolysis was sigmoidalwith a transition at pH 4.5, which was identical with the experimentallydetermined pK_a of Glu35 in the covalent adduct, indicating thatGlu35 functions as a general base to hydrolyze the adduct. Toimprove the turnover rate of D52E lysozyme, the mutation ofN46D was designed and introduced to D52E lysozyme. This mutationreduced the activation energy in the hydrolysis reaction ofthe covalent adduct by 1.8 kcal/mol at pH 5.0 and 40°C butdid not affect the formation reaction. Our data may providea useful approach to understanding the precise mechanism ofthe function of natural glycosidases, which catalyze via a covalentadduct.     

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.     

Improved peptide function from random mutagenesis over short 'windows'

We have applied random mutagenesis over short contiguous residuetracts (‘windows’) within an active peptide (the-peptide of ß-galactosidase) such that all windowresidues are replaced simultaneously. A novel technique usingmixed synthetic oligonucleotides and selection against an EcoKrestrictionsite has allowed the construction of libraries of mutants fortwo separate windows, sites A and B. Mutant phenotypes can beeasily assessed in vivoby a complementation test, and panelsof mutants have been quantitatively tested in vivoThis allowedthe rapid probing of structural requirements for each site.The two windows yielded markedly disparate results. Site B wasmuch less stringent in its sequence requirements for significantfunction than Site A, and mutants with improved function wereisolated at Site B alone. In addition, one Site B mutant withwild-type levels of activity showed enhanced stability to heator a protein denaturant. We propose that short tracts with thecharacteristics of Site B constitute ‘secondary’interaction sites which are more tolerant of sequence diversity.Random manipulation of such secondary sites is thus more likelyto yield up-mutations for standard or altered environments.Window mutagenesis can in principle be applied to any protein-proteinor protein-Ugand interaction.     

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     

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.     

Site-directed mutagenesis of Arg60 and Cys271 in ornithine transcarbamylase from rat liver

We have investigated the putative carbamylphosphate- and ornithine-bindingdomains in ornithine transcarbamylase from rat liver using site-directedmutagenesis. Arg60, present in the phosphate-binding motif X-Ser-X-Arg-Xand therefore implicated in the binding of the phosphate moietyof carbamylphosphate has been replaced with a leucine. Thisresults in a dramatic reduction of catalytic activity, althoughthe enzyme is synthesized in cells stably transfected with themutant clone and imported, correctly processed and assembledinto a homotrimer in mitochondria. The sole cysteine residue(Cys271) has been implicated in ornithine binding by the chemicalmodification studies of Marshall and Cohen in 1972 and 1980(J. Biol. Chem., 247, 1654–1668, 1669–1682; 255,7291–7295, 7296–7300). Replacement of this residuewith serine did not eliminate enzyme activity but affected theMichaelis constant for ornithine (K_b, increasing it 5-fold from0.71 to 3.7 mM and reduced the k_cat at pH 8.5 by 20-fold. Thesechanges represent a loss in apparent binding energy for theenzyme - ornithine complex of 2.9 kcal/mol, suggesting thatCys271 is normally involved in hydrogen bonding to the substrate,ornithine. The cysteine to serine substitution also caused thedissociation constant (Kä for the competitive inhibitor,L-norvaline to be increased 10-fold, from 12 to 120 µM.The small loss in binding energy and relatively high residualcatalytic activity of the mutant strongly suggests that a numberof other residues are involved in the binding of ornithine.The effect of replacement of Cys271 with serine was restrictedto the ornithine binding site of the enzyme since both the bindingconstant for carbamyl-phosphate (K_ia) and Michaelis constant(K_a) were not appreciably different for mutant and wild-typeenzymes. The pH optimum of the wild-type enzyme (8.6) is increasedto > 9.6 in the Ser271 mutant.     

Breakdown in the relationship between thermal and thermodynamic stability in an interleukin-1{beta} point mutant modified in a surface loop

Sequence variants of the ß-barrel protein interleukin-1ßhave been analyzed for their stabilities toward irreversiblethermal inactivation by monitoring the generation of light scatteringaggregates on heating. The derived temperatures for the onsetof aggregation (T_agg values) correlate well with the free energiesof unfolding of these proteins with the exception of one variant,Lys97—Val (K97V), which undergoes aggregation at a temperature7°C lower than expected based on its thermodynamic stability.This lower than expected thermal stability may be due to generationof an aggregation-prone unfolding intermediate at a temperaturelower than the T_m of the global transition. This hypothesisis supported by the location of residue 97 in the long 86–99loop which has structural features suggesting it may comprisea small, independent folding unit or microdomain. The excellentcorrelation of thermal and thermodynamic stabilities of sevenof the eight variants tested is consistent with accepted modelsfor thermal inactivation of proteins. At the same time the poorfit of the K97V variant underscores the risk in using thermalstability data in quantitative analysis of mutational studiesof the folding stability of proteins.     

A thermoresistant mutant of ribonuclease T1 having three disulfide bonds

Molecular-dynamic calculations predict that, if Tyr24 and Asn84are each replaced by a Cys residue, it should be possible toform a third disulfide bond in ribonuclease T1 (RNase T1) betweenthese residues, with only minimal conformational changes atthe catalytic site. The gene encoding such a mutant variantof RNase T1 (Tyr24 – Cys24, Asn84 – Cys84) was constructedby the cassette mutagenesis method using a chemically synthesizedgene. In order to reduce the toxic effect of the mutant enzyme(RNase T1S) on an Escherichia coli host, we arranged for theprotein to be secreted into the periplasmic space by using avector that harbors a gene for an alkaline phosphatase signalpeptide under the control of the trp promoter. The nucleolyticactivity of RNase T1S toward pGpC was approximately the sameas that of RNase T1 at 37°C (pH 7.5). Moreover, at 55°C,RNase T1S retained nearly 70% of its activity while the activityof the wild-type enzyme was reduced to <10%. RNase T1S wasalso more resistant to denaturation by urea than the wild-typeenzyme. However, unlike RNase T1, RNase T1S was irreversiblyand almost totally inactivated by boiling at 100°C for 15min.     

Modular mutagenesis of a TEM-barrel enzyme: the crystal structure of a chimeric E.coli TIM having the eighth {beta}{alpha}-unit replaced by the equivalent unit of chicken TIM

Abstract The crystal structure of a hybrid Escherichia coli triosephosphateisomerase (TIM) has been determined at 2.8 Å resolution.The hybrid TIM (ETIM8CHI) was constructed by replacing the eighthß-unit of E.coli TIM with the equivalent unit of chickenTIM. This replacement involves 10 sequence changes. One of thechanges concerns the mutation of a buried alanine (Ala232 instrand 8) into a phenylalanine. The ETIM8CHI structure showsthat the A232F sequence change can be incorporated by a side-chainrotation of Phe224 (in helix 7). No cavities or strained dihedralsare observed in ETIM8CHI in the region near position 232, whichis in agreement with the observation that ETIM8CHI and E.coliTIM have similar stabilities. The largest CA (C-alpha atom)movements, 3 Å, are seen for the C-terminal end of helix8 (associated with the outward rotation of Phe224) and for theresidues in the loop after helix 1 (associated with sequencechanges in helix 8). From the structure it is not clear whythe k_cat of ETIM8CHI is 10 times lower than in wild type E.coliTIM     

Effective renaturation of reduced lysozyme by gentle removal of urea

To increase the folding yield of concentrated reduced lysozyme,we developed a renaturation method by means of dialysis fromconcentrated urea with redox agents. After lysozyme was incubatedin the reducing buffer (8 M urea solution) with oxidized glutathione,renaturation of reduced lysozyme was started by dialysis againstthe dialyzing buffer containing 8 M urea with redox agents.The urea concentration of the dialyzing bottle was graduallydiluted with dialyzing buffer without urea at a flow rate of0.1 ml/min by high pressure pump. Using this systematic dialysis,a concentration as high as 5 mg/ml of reduced lysozyme couldbe renaturated in 80% yield, while the folding yield was <5%even at a concentration of 1 mg/ml using a conventional rapiddilution method [Goldberg et al. (1991) Biochemistry, 30, 2790–2797].Therefore, it was concluded that gentle removal of urea fromdenatured proteins, dissolved in concentrated urea solution,by means of dialysis should be useful to renature denaturedproteins effectively.     

Mutagenesis of conserved residues within the active site of Escherichia coli alkaline phosphatase yields enzymes with increased kcat

The likelihood for improvement in the catalytic properties ofEscherichia coli alkaline phosphatase was examined using site-directedmutagenesis. Mutants were constructed by introducing sequencechanges into nine preselected amino acid sites within 10 A ofthe catalytic residue serine 102. When highly conserved residuesin the family of alkaline phosphatases were mutated, many ofthe resulting enzymes not only maintained activity, but alsoexhibited greatly improved tra,. Of –170 mutant enzymesscreened, 5% (eight mutants) exhibited significant increasesin specific activity. In particular, a substitution by serineof a totally invariant AsplOl resulted in a 35-fold increaseof specific activity over wild-type at pH 10.0. Up to 6-foldincreases the k_cat/k_m ratio were observed.     

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.     

Determination of the pKa values of titratable groups of an antigen-antibody complex, HyHEL-5-hen egg lysozyme

The titration behavior of the ionizable residues of the HyHEL-5–henegg lysozyme complex and its individual components has beenstudied using continuum electrostatic calculations. Severalresidues of HyHEL-5 had pK_a values shifted away from model valuesfor isolated residues by more than three pH units. Shifts awayfrom the model values were smaller for the residues of hen egglysozyme. A moderate variation in the pK_a values of the titratablegroups was observed upon increase of the ionic strength from0 to 100 mM, amounting to 1–2 pH units in most cases.Under physiological conditions, the net charge of HyHEL-5 wasopposite that for hen egg lysozyme. Several residues, includingthose involved in the Arg–Glu salt bridges that have beenproposed to be important in antibody-antigen binding, had pK_avalues that were changed significantly upon binding. The maintitration event upon antibody-antigen binding appears to beloss of a proton from residue GluH50 of the Fv molecule. Thelimitations of our calculation methods and the role they mightplay in the design of antibodies for use in assays, sensorsand separations are discussed