Protein engineering of alcohol dehydrogenase -- 1. Effects of two amino acid changes in the active site of yeast ADH-1

One of the promises held out by protein engineering is the abilityto alter predictably the properties of an enzyme to enable itto find new substrates or catalyse existing substrates moreefficiently, such manipulations being of interest both enzymologicallyand, potentially, industrially. It has been postulated thatin yeast alcohol dehydrogenase (YADH-1) certain amino acidssuch as Trp 93 and Thr 48 constrict the active site due to theirbulky side chains and thus impede catalysis of molecules largerthan ethanol. To study effects of enlarging the active sitewe have made two changes into YADH-1, replacing Trp 93 withPhe and Thr 48 with Ser. Kinetic experiments showed that thisenzyme had marked increases in reaction velocity for the n-alcoholspropanol, butanol, pentanol, hexanol, heptanol, octanol andcinnamyl alcohol compared to the parent, agreeing with the predictionthat expanding the active site should facilitate the oxidationof larger alcohols. The substrate affinities were slightly reducedin the altered enzyme, possibly due to its having reduced hydrophobicityat Phe 93.     

Engineering yeast alcohol dehydrogenase. Replacing Trp54 by Leu broadens substrate specificity

Analysis of a crystal structure of alcohol dehydrogenase (Adh)from horse liver suggests that Trp54 in the homologous yeastalcohol dehydrogenase prevents the yeast enzyme from efficientlycatalysing the oxidation of long-chain primary alcohols withbranching at the 4 position (e.g. 4-methyl-1-pentanol, cinnamylalcohol). This residue has been altered to Leu by site-directedmutagenesis. The alteration yields an enzyme that serves asan effective catalyst for both longer straight-chain primaryalcohols and branched chain alcohols.     

Single amino acid substitutions can further increase the stability of a thermophilic L-lactate dehydrogenase

Lactate dehydrogenases are of considerable interest as stereospecificcatalysts in the chemical preparation of enantiomerically pure-hydroxyacid synthons. For such applications in synthetic organicchemistry it would be desirable to have enzymes which tolerateelevated temperatures for prolonged reaction times, to increaseproductivity and to extend then applicability to poor substrates.Here, two examples are reported of significant thermostabilizations,induced by sitedirected mutagenesis, of an already thermostableprotein, the L-lactate dehydrogenase (EC 1.1.1.27 [EC] , 35 kDa permonomer subunit) from Bacillus stearothermophilus. Thermal inactivationof this enzyme is accompanied by irreversible unfolding of thenative protein structure. The replacement of Argl71 by Tyr stabilizesthe enzyme against thermal inactivation and unfolding. Thisstabilizing effect appears to be based on improved interactionsbetween the subunits in the core of the active dimeric or tetramericforms of the enzyme. The thermal stability of L-lactate dehydrogenasevariants with an active site Arg residue, either in the 171(wild-type) or in the 102 position, is further increased bysulfate ions. The two stabilizing effects are additive, as foundfor the Argl71Tyr/ Gln1O2Arg double mutant, for which the stabilityof the protein in 100 mM sulfate solution reaches that of L-lactatedehydrogenases from extreme thermophiles. All mutant proteinsretain significant catalytic activity, both in the presenceand absence of stnhilfoing salts, and are viable catalysts inpreparative scale reactions.     

Alteration of the amino acid substrate specificity of clostridial glutamate dehydrogenase by site-directed mutagenesis of an active-site lysine residue

Two residues, K89 and S380, thought to interact with the -carboxylgroup of the substrate L-glutamate, have been altered by site-directedmutagenesis of clostridial glutamate dehydrogenase (GDH). Thesingle mutants K89L and S380V and the combined double mutantK89L/S380V were constructed. All three mutants were satisfactorilyoverproduced in soluble form. However, only the K89L mutantwas retained by the dye column normally used in purifying thewild-type enzyme. All three mutant enzymes were purified tohomogeneity and tested for substrate specificity with 24 aminoacids. The single mutant S380V showed no detectable activity.The alternative single mutant K89L showed an activity towardsL-glutamate that was decreased nearly 2000-fold compared withwild-type enzyme, whereas the activities towards the monocarboxylicsubstrates -aminobutyrate and norvaline were increased 2- to3-fold. A similar level of activity was obtained with methionine(0.005 U/mg) and norleucine (0.012 U/mg), neither of which giveany activity with the wild-type enzyme under the same conditions.The double mutant showed decreased activity with all substratescompared with the wild-type GDH. In view of its novel activities,the K89L mutant was investigated in greater detail. A strictlylinear relationship between reaction velocity and substrateconcentration was observed up to 80 mM L-methionine and 200mM L-norleucine, implying very high K_m values. Values of k_cat/K_m,for L-methionine and L-norleucine were 6.7x10^–2 and 0.15s^–1M^–1, respectively. Measurements with dithiobisnitrobenzoicacid showed that the mutant enzymes all reacted with a stoichiometryof one -SH group per subunit and all showed protection by coenzyme,indicating essentially unimpaired coenzyme binding. With glutamateor 2-oxoglutarate as substrate the K_m values for the vestigialactivity in the mutant enzyme preparations were strikingly closeto the wild-type K_m values. Both for wild-type GDH and K89L,L-glutamate gave competitive product inhibition of 2-oxoglutaratereduction but did not inhibit the reduction of 2-oxocaproatecatalysed by K89L enzyme. This suggests that the low levelsof glutamate/2-oxoglutarate activity shown by the mutant enzymeare due to trace contamination. Since stringent precautionswere taken, it appears possible that this reflects the levelof reading error during overexpression of the mutant proteins.CD measurements indicate that the S380V mutant has an alteredconformation, whereas the K89L enzyme gave an identical CD spectrumto that of wild-type GDH; the spectrum of the double mutantwas similar, although somewhat altered in intensity. The resultsconfirm the key role of K89 in dicarboxylate recognition byGDH.     

Three-dimensional structures of chimeric enzymes between Bacillus subtilis and Thermus thermophilus 3-isopropylmalate dehydrogenases

The 3-D structures of two chimeric enzymes (4M6T and 2T2M6T)between the Bacillus subtilis and Thermus thermophilus 3-isopropylmalatedehydrogenases were analysed by X-ray diffraction in order toinvestigate their different thermostabilities. The structureof 2T2M6T was determined by the difference Fourier method andthat of 4M6T by rigid body refinement, as based on the structureof the T. thermophilus enzyme. These structures were refinedstereochemically to an R-factor of 0.193 at 2.5 Å resolutionfor 4M6T and to an R-factor of 0.195 at 2.2 Å resolutionfor 2T2M6T. The 3-D structures of 4M6T and 2T2M6T were veryclose to the structure of the T.thermophilus enzyme, conspicuousdifferences being at the molecular surface, In particular, 2T2M6Thaving a larger reduction in thermostability was more closelyrelated to the T.thermophilus enzyme. However, their correlationsbetween C-atom displacements and the root squares of the temperaturefactors were significantly different from each other.     

Contribution of amino acid substitutions at two different interior positions to the conformational stability of human lysozyme

To elucidate correlative relationships between structural changeand thermodynamic stability in proteins, a series of mutanthuman lysozymes modified at two buried positions (Ile56 andIle59) were examined. Their thermodynamic parameters of denaturationand crystal structures were studied by calorimetry and X-raycrystallography. The mutants at positions 56 and 59 exhibiteddifferent responses to a series of amino acid substitutions.The changes in stability due to substitutions showed a linearcorrelation with changes in hydrophobicity of substituted residues,having different slopes at each mutation site. However, thestability of each mutant was found to be represented by a uniqueequation involving physical properties calculated from mutantstructures. By fitting present and previous stability data formutant human lysozymes substituted at various positions to theequation, the magnitudes of the hydrophobicity of a carbon atomand the hydrophobicity of nitrogen and neutral oxygen atomswere found to be 0.178 and –0.013 kJ/mol.Ų, respectively.It was also found that the contribution of a hydrogen bond witha length of 3.0 Å to protein stability was 5.1 kJ/moland the entropy loss of newly introduction of a water moleculeswas 7.8 kJ/mol.     

Protein engineering of cytochrome c by semisynthesis: substitutions at Glutamic acid 66

We have used protein semisynthesis to prepare four analoguesof horse cytochrome c, in which the glutamic acid residue atposition 66 has been removed and replaced by norvaline, glutamine,lysine and, as a methodological control, glutamic acid. Thisresidue is quite strongly conserved in mitochondrial cytochromec, and forms part of a cluster of acidic residues that occursin all cytochromes c but whose function is obscure. Comparativestudies of the physical and biochemical properties of the analogueshave now disclosed two specific roles for Glu⁶⁶ in the protein.It contributes significantly to the stabilization of the activeconformation of the protein, probably by salt bridge formation,and it appears to participate in the redox-state-dependent ATP-bindingsite of cytochrome c. Our results also support two general viewsof the role of surface charged residues in cytochrome c, namelythat their disposition influences both redox potential, throughthe electrostatic field felt at the redox centre, and the kineticsof electron transfer, through the dipole moment they generate.     

Replacing the glutamate ligand in the structural zinc site of Sulfolobus solfataricus alcohol dehydrogenase with a cysteine decreases thermostability

The alcohol dehydrogenase gene from the thermophilic archaeumSulfolobus solfataricus has been subcloned and expressed inEscherichia coli under the control of the T7 inducible promoter.Therecombinant protein shows properties analogous to those of thenative enzyme, including thermostability, despite the fact thatE.coli does not post-translationally modify two lysine residueswhich are N--methylated in the native enzyme. We constructeda 3-D model of the S.solfataricus alcohol dehydrogenase usingthe known structure of its isozyme from horse liver as a template.Our analysis of the structural zinc binding site suggested thatthis site is present andfunctional in the S.solfataricus enzymeand that a glutamate ligand can contribute to thermostabilityby influencing electrostatic interactions around the metal centre.To investigate thishypothesis, we constructed, expressed andcharacterized a mutant where the glutamate is replaced by acysteine, thus restoring the zinc binding site of mesophilicalcohol dehydrogenases. Themutant shows the same activity buta reduced thermostability with respect to the wild-type recombinantprotein, as suggested by our model.     

Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K

The catalytic contribution of His48 in the active site of porcinepancreatic phospholipase A2 was examined using site-directedmutagenesis. Replacement of His48 by lysine (H48K) gives riseto a protein having a distorted lipid binding pocket. Activityof this variant drops below the detection limit which is 10⁷-foldlower than that of the wild-type enzyme. On the other hand,the presence of glutamine (H48Q) or asparagine (H48N) at thisposition does not affect the structural integrity of the enzymeas can be derived from the preserved lipid binding propertiesof these variants. However, the substitutions H48Q and H48Nstrongly reduce the turnover number, i.e. by a factor of 10⁵.Residual activity is totally lost after addition of a competitiveinhibitor. We conclude that proper lipid binding on its ownaccelerates ester bond hydrolysis by a factor of 10². With theselected variants, we were also able to dissect the contributionof the hydrogen bond between Asp99 and His48 on conformationalstability, being 5.2 kJ/mol. Another hydrogen bond with His48is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycolinteracts with the enzyme. Its contribution to binding of theinhibitor in the presence of an interface was found to be 5.7kJ/mol.     

Synthesis of a squash-type protease inhibitor by gene engineering and effects of replacements of conserved hydrophobic amino acid residues on its inhibitory activity

Cucurbita maxima trypsin inhibitor I (CMTI-I), a member of thesquash-type protease inhibitor family, is composed of 29 aminoacids and shows strong inhibition of trypsin by its compactstructure. To study the structure–function relationshipof this inhibitor using protein engineering methods, we constructedan expression system for CMTI-I as a fused protein with porcineadenylate kinase (ADK). A Met residue was introduced into thejunction of ADK and CMTI-I to cleave the fusion protein withCNBr, whereas a Met at position 8 of authentic CMTI-I was replacedby Leu. Escherichia coli JM109 transformed with the constructedplasmid expressed the fused protein as an inclusion body. Aftercleavage of the expressed protein with CNBr, fully reduced speciesof CMTI-I were purified by reversed-phase HPLC and then oxidizedwith air by shaking. For efficient refolding of CMTI-I, we used50 mM NH₄HCO₃ (pH 7.8) containing 0.1% PEG 6000 at higher proteinconcentration. Strong inhibitory activity toward trypsin wasdetected only in the first of three HPLC peaks. The inhibitorconstant of CMTI-I thus obtained, in which Met8 was replacedby Leu, was 1.4x10-¹⁰ M. The effect of replacement of Met withLeu at position 8 was shown to be small by comparison of theinhibitor constant of authentic CMTI-III bearing Lys at position9 (8.9x10-¹¹ M) with that of its mutant bearing Leu at position8 and Lys at position 9 (1.8x10-¹⁰ M). To investigate the roleof the well conserved hydrophobic residues of CMTI-I in itsinteraction with trypsin, CMTI-I mutants in which one or allof the four hydrophobic residues were replaced by Ala were prepared.The inhibitor constants of these mutants indicated that thosewith single replacements were 5–40 times less effectiveas trypsin inhibitors and that the quadruple mutant was –450times less effective, suggesting that the hydrophobic residuesin CMTI-I contribute to its tight binding with trypsin. However,each mutant was not converted to a temporary inhibitor.     

Protein engineering of chymosin; modification of the optimum pH of enzyme catalysis

The aspartic proteinase chymosin exhibits a local network ofhydrogen bonds involving the active site aspartates and surroundingresidues which may have an influence on the rate and optimalpH of substrate cleavage. We have introduced into chymosin Bthe following substitutions: Asp304 to Ala (D304A), Thr218 toAla (T218A) and Gly244 to Asp (G244D, chymosin A), using oligonucleotide-directedmutagenesis. Kinetic analysis of these active mutants showsshifts in their pH optima to 4.4 D304A, 4.2 T218A and 4.0 G244Dcompared with 3.8 for chymosin B using a synthetic octapeptidesubstrate. The upward shift of the D304A and T218A may be dueto the loss of hydrogen bond interactions indirectly affectingthe catalytic aspartates 32 and 215. The G244D mutation whichis in a flexible loop on the surface of the enzyme may alterthe conformation of the specificity pockets on the prime sideof the scissile bond.     

Exchange of domains of glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus and the mesophilic bacterium Clostridium difficile: effects on catalysis, thermoactivity and stability

The glutamate dehydrogenase gene from the hyperthermophilicarchaeon Pyrococcus furiosus has been functionally expressedin Escherichia coli under the control of the X, PL promoter.The P.furiosus glutamate dehydrogenase amounted to 20% of thetotal E.coli cell protein, and the vast majority consisted ofhexamers. Following activation by heat treatment, an enzymecould be purified from E.coli that was indistinguishable fromthe glutamate dehydrogenase purified from P.furiosus. Hybridgenes, that consisted of the coding regions for the homologousglutamate dehydrogenases from P.furiosus and the mesophilicbacterium Clostridium difficile, were constructed and successfullyexpressed in E.coli. One of the resulting hybrid proteins, containingthe glutamate binding domain of the C.difficile enzyme and thecofactor binding domain of the P.furiosus enzyme, did not showa detectable activity. In contrast, the complementary hybridcontaining the P.furiosus glutamate and the C.difficile cofactorbinding domain was a catalytically active hexamer that showeda reduced substrate affinity but maintained efficient cofactorbinding with the specificity found in the Clostridium symbiosumenzyme. Compared with the C.difficile glutamate dehydrogenase,the archaeal-bacterial hybrid is slightly more thermoactive,less thermostable but much more stable towards guanidinium chloride-inducedinactivation and denaturation     

Protein fragment variability analysis and some principles of protein engineering of vaccines

Based on protein sequence databank (PIR), the ‘variablefragment’ bank, comprising pairs of closely-related proteins,containing one or more strongly differing sites of primary structures,was formed. The bank includes 465 ‘variable fragments’in 383 protein pairs. The amino acid composition of ‘variablefragments’ was examined and indices of potential aminoacid residue variability were formed. An analysis of the interchangeabilityof amino acid fragments depending on the substitution site (N-or C-terminal, or middle part of a chain), the fragment lengthdifferences and physico-chemical properties of residues, suchas volume, hydrophobicity, polarity and isoelectric point, wascarried out. Based on this analysis some general empirical rulesof peptide insertions in carrier proteins were created. Therules are directed at performing modifications leaving the generalstructure and function of the carrier protein molecule unchanged.The selection scheme for the regions suitable for modificationand the criteria for determination of the range of acceptablevariations in these regions were suggested. The use of the potentialvariability profile for detecting regions suitable for peptideinsertion was considered using surface protein of hepatitisB virus as an example.     

Improving protein solubility through rationally designed amino acid replacements: solubilization of the trimethoprim-resistant type S1 dihydrofolate reductase

In recent years resistance to the antibacterial agent trimethoprim(Tmp) has become more widespread and several Tmp-resistant (Tmp^rdihydrofolate reductases (DHFRs) have been described from Gram-negativebacteria. In staphylococci, however, only one Tmp^r DHFR (typeS1 DHFR) has been found so far, and this is located on transposonTn4003. To help understand the mechanism of resistance, we areinterested in determining the 3-D structure of the recombinantenzyme produced in Escherichia coli. However, the productionlevel of the type S1 DHFR was very low and >95% of the totalrecombinant protein accumulated in inclusion bodies. Furthermore,as a result of an internal start of translation, a truncatedderivative of the enzyme that copurified with the full-lengthenzyme was produced. We were able to increase the expressionlevel 20-fold by changing 18 N-terminal codons and to eliminatethe internal start of translation. In addition, through molecularmodelling and subsequent site-directed mutagenesis to replacetwo amino acids, we constructed a biochemically similar butsoluble derivative of the type SI DHFR that, after productionin E.coli, resulted in a 264-fold increase in DHFR activity.The highly overproduced enzyme was purified to homogeneity,characterized biochemically and crystallized.     

Site-directed mutagenesis of aspartic acid 372 at the ATP binding site of yeast phosphoglycerate kinase: over-expression and characterization of the mutant enzyme

A new phosphoglycerate kinase over-expression vector, pYE-PGK,has been constructed which greatly facilitates the insertionand removal of mutant enzyme genes by cleavage at newly introducedBamtHI sites. This vector has been used to prepare mutant proteinin appreciable (100 mg) quantities for use in kinetic, crystaUographicand NMR experiments. Aspartate 372 is an invariant amino acidresidue in genes known to code for a functionally active PGK.The function of this acidic residue appears to be to help desolvatethe magnesium ion compfexed with either ADP or ATP when thissubstrate binds to the enzyme. Both crystallographk and nuclearmagnetic resonance experiments show that the replacement ofthe residue with asparagine has only minimal effects on theoverall structure. The substitution of the charged carboxylgroup with that of the neutral amide affects the binding ofthe nucleotide substrate as predicted but not, as might havebeen expected, the binding of 3-phospho-glycerate. The overallvelocity of the enzymic reaction (V_max) is reduced 10-fold bythe substitution of aspartic acid 372 by an asparagine residue(D372N). This reduction in V_max is considerably less than onewould expect from its known position within the structure ofthe enzyme. This result therefore poses questions about ourunderstanding of charged groups at the active centres of enzymesand of the reason for their apparent conservation.     

Analysis of amino acid indices and mutation matrices for sequence comparison and structure prediction of proteins

An amino acid index is a set of 20 numerical values representingany of the different physicochemical and biochemical propertiesof amino adds. As a follow-up to the previous study, we haveincreased the size of the database, which currently contains402 published indices, and re-performed the single-linkage clusteranalysis. The results basically confirmed the previous findings.Another important feature of amino acids that can be representednumerically is the similarity between them. Thus, a similaritymatrix, also called a mutation matrix, is a set of 20x20 numericalvalues used for protein sequence alignments and similarity searches.We have collected 42 published matrices, performed hierarchicalcluster analyses and identified several clusters correspondingto the nature of the data set and the method used for constructingthe mutation matrix. Further, we have tried to reproduce eachmutation matrix by the combination of amino acid indices inorder to understand which properties of amino acids are reflectedmost. There was a relationship between the PAM units of Dayhoff'smutation matrix and the volume and hydrophobicity of amino adds.The database of 402 amino acid indices and 42 amino acid mutationmatrices is made publicly available on the Internet.     

Redesign of the coenzyme specificity in L-Lactate dehydrogenase from Bacillus stearothermophilus using site-directed mutagenesis and media engineering

L-Lactate dehydrogenase (LDH) from Bacillus stearothermophilusis a redox enzyme which has a strong preference for NADH overNADPH as coenzyme. To exclude NADPH from the coenzyme-bindingpocket, LDH contains a conserved aspartate residue at position52. However, this residue is probably not solely responsiblefor the NADH specificity. In this report we examine the possibilitiesof altering the coenzyme specificity of LDH by introducing arange of different point mutations in the coenzyme-binding domain.Furthermore, after choosing the mutant with the highest selectivityfor NADPH, we also investigated the possibility of further alteringthe coenzyme specificity by adding an organic solvent to thereaction mixture. The LDH mutant, I51K:D52S, exhibited a 56-foldincreased specificity to NADPH over the wild-type LDH in a reactionmixture containing 15% methanol. Furthermore, the NADPH turnovernumber of this mutant was increased almost fourfold as comparedwith wild-type LDH. To explain the altered coenzyme specificityexhibited by the D52SI51K double mutant, molecular dynamicssimulations were performed.     

Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocket

Serine endoproteases such as trypsins and subtilisins are knownto have an extended substrate binding region that interactswith residues P6 to P3' of a substrate. In order to investigatethe structural and functional effects of replacing residuesat the S4 substrate binding pocket, the serine protease fromthe alkalophilic Bacillus strain PB92, which shows homologywith the subtilisins, was mutated at positions 102 and 126–128.Substitution of Val102 by Trp results in a 12–fold increasein activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide(sAAPFpNA). An X-ray structure analysis of the V102W mutantshows that the Trp side chain occupies a hydrophobic pocketat the surface of the molecule leaving a narrow crevice forthe P4 residue of a substrate. Better binding of sAAPFpNA bythe mutant compared with the wild type protein as indicatedby the kinetic data might be due to the hydrophobic interactionof Ala P4 of the substrate with the introduced Trp102 side chain.The observed difference in binding of sAAPFpNA by protease PB92and thermitase, both of which possess a Trp at position 102,is probably related to the amino acid substitutions at positions105 and 126 (in the protease PB92 numbering).Kinetic data forthe variants obtained by random mutation of residues Serl26,Prol27 and Serl28 reveal that the activity towards sAAPFpNAincreases when a hydrophobic residue is introduced at position126. An X-ray diffraction analysis was carried out for the threeprotease PB92 mutants which have residues Serl26-Prol27-Serl28replaced by Met-Ala-Gly(‘MAG’ mutant), Phe-Gln-Ser(‘FQS’ mutant) and Asn-Ser-Ala (‘NSA’mutant). Met 126 and Phel26 in the crystal structures of thecorresponding mutants are fixed in the same hydrophobic environmentas Trp102 in the V102W mutant.In contrast, Asnl26 in the ‘NSA’mutant is completely disordered in both crystal forms for whichthe structure has been determined. According to our kineticmeasurements none of the mutants with Met, Phe, Leu or Val atposition 126 binds sAAPFpNA better than the wild type enzyme.Resultsof the site-directed mutagenesis at position 127 imply thatpossible interaction of this residue with a substrate has almostno effect on activity towards sAAPFpNA and casein.     

Effects of hydrophobic amino acid substitution in Pleurotus ostreatus proteinase A inhibitor 1 on its structure and functions as protease inhibitor and intramolecular chaperone

We previously demonstrated that Pleurotus ostreatus proteinase A inhibitor 1 (POIA1) could function as an intramolecular chaperone of subtilisin BPN', as in the case of the propeptide of subtilisin BPN', and that its Phe44 --> Ala mutant, which lost its tertiary structure, could not assist the refolding of subtilisin BPN'. In this study, we examined the effects of hydrophobic amino acid substitutions at other sites and substitutions of Phe44 with other hydrophobic residues on the structure and functions of POIA1. These mutations were introduced into POIA1cm that had been obtained by the substitution of the C-terminal six residues of POIA1 with those of the propeptide of subtilisin BPN'. When Ile32 or Ile64 was substituted with Ala, the tertiary structure of the resultant mutant was markedly destroyed, and the activities as a protease inhibitor and an intramolecular chaperone were significantly lowered. Among the position 44 mutants, the Phe44 --> Val mutant was a much less effective intramolecular chaperone with conversion to a digestible inhibitor, possibly owing to destruction of the tertiary structure. On the other hand, the Phe44 --> Leu or Ile mutant maintained its tertiary structure, and hence could function as a more effective intramolecular chaperone than the Phe44 --> Val mutant. Furthermore, since the Phe44 --> Leu mutant was a more susceptible inhibitor than POIA1cm, the halo formed around a colony of Bacillus cells transformed with a plasmid encoding this mutant was larger than others. These results clearly show the close relationship between the tertiary structure and functions of POIA1 as a protease inhibitor and an intramolecular chaperone, and that a combination of such inhibitory properties and intramolecular chaperone activity of POIA1 might affect the diameter of the halo formed around Bacillus colonies in vivo.     

Prediction of signal peptide functional properties: a study of the orientation and angle of insertion of yeast invertase mutants and human apolipoprotein B signal peptide variants

A number of studies have introduced mutations into the yeastinvertase signal peptide, using it as a model system to elucidatefeatures for targeting, translocation and intracellular transportUsing molecular modelling of the invertase signal peptide wehave analysed the hydrophobicity potential and the change inthe dielectric constant of the energy transfer, when the moleculemoves from a hydrophobic to a hydrophilic phase at the simulatedhydrophobic-hydrophilic interface. This modelling has been carriedout on wild type and mutant invertase signal peptides of alteredfunction, previously reported in the literature. While the predictedangle of insertion correlates with the measured extent of invertasesecretion, with an optimum angle of 45, mutations that changethe angle of orientation reduce the extent of invertase secretion.We have applied these same molecular modelling principles tothe naturally occurring variants of the human apolipo-proteinB (apoB) signal peptide, that confer a secretion defective phenotypewhen fused to yeast invertase and expressed in yeast. Our modellingthus identifies a strong correlation between the predicted angleof insertion of the signal peptide into the membrane and itsability to direct secretion.