Analysis of structural determinants of the stability of thermolysinlike proteases by molecular modelling and site-directed mutagenesis

The thermolysin-like protease (TLP) produced by Bacillus stearothermophilusCU21 (TLP-ste) differs at 43 positions from the more thermallystable thermolysin (containing 316 residues in total). Of thesedifferences, 26 were analysed by studying the effect of replacingresidues in TLP-ste by the corresponding residues in thermolysin.Several stabilizing mutations were identified but, remarkably,considerable destabilizing mutational effects were also found.A Tyr-rich three residue insertion in TLP-ste (the only deletional/insertionaldifference between the two enzymes) appeared to make an importantcontribution to the stability of the enzyme. Mutations withlarge effects on stability were all localized in the ßpleatedN-terminal domain of TLP-ste, confirming observations that thisdomain has a lower intrinsic stability than the largely -helicalC-terminal domain. Rigidifying mutations such as Gly58 Alaand Ala69 Pro were among the most stabilizing ones. Apart fromthis observation, the analyses did not reveal general rulesfor stabilizing proteins. Instead, the results highlight theimportance of context in evaluating the stability effects ofmutations.     

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.     

Conformational stability of human skeletal tropomyosins modified by site-directed mutagenesis

We have used human ß-tropomyosin produced in Escherichiacoli and deletion mutants obtained by site-directed mutagenesisto analyse the conformational stability of this molecule undervarious experimental conditions. Protein engineering has allowedus to answer some questions raised by stability analysis ofthe wild-type tropomyosin. The complex pattern of denaturationis due neither to heterogeneity of the preparation nor to head-to-tailinteractions. The N- and C-termini are not of importance forthe thermal stability of the molecule. On the contrary, deletionof the 31 C-terminus amino acids leads to a dramatic decreaseof the stability observed in guanidinium chloride. This loweringis interpreted as the participation of one more guanidiniumchloride ions to the denaturation equilibrium. Analysis of thestability in presence of organic solvents reveals that acetonitrileand methanol induce opposite effects. Investigation of theseeffects by three methods (CD, fluorescence and electrophoresisthat measure respectively the content in alpha-helix, the contactbetween the two strands and the strands exchange) leads to theconclusion that strand separation can precede the denaturationof the alpha-helix.     

Molecular modelling and site-directed mutagenesis of human GALR1 galanin receptor defines determinants of receptor subtype specificity

Human galanin is a 30 amino acid neuropeptide that elicits arange of biological activities by interaction with G protein-coupledreceptors. We have generated a model of the human GALR1 galaninreceptor subtype (hGALR1) based on the alpha carbon maps offrog rhodopsin and investigated the significance of potentialcontact residues suggested by the model using site-directedmutagenesis. Mutation of Phe186 within the second extracellularloop to Ala resulted in a 6-fold decrease in affinity for galanin,representing a change in free energy consistent with hydrophobicinteraction. Our model suggests interaction between Phe186 ofhGALR1 and Ala7 or Leu11 of galanin. Receptor subtype specificitywas investigated by replacement of residues in hGALR1 with thecorresponding residues in hGALR2 and use of the hGALR2-specificligands hGalanin(2–30) and [D-Trp2]hGalanin(1–30).The His267Ile mutant receptor exhibited a pharmacological profilecorresponding to that of hGALR1, suggesting that His267 is notinvolved in a receptor–ligand interaction. The mutationPhe115Ala resulted in a decreased binding affinity for hGalaninand for hGALR2-specific analogues, indicating Phe115 to be ofstructural importance to the ligand binding pocket of hGALR1but not involved in direct ligand interaction. Analysis of Glu271Trpsuggested that Glu271 of hGALR1 interacts with the N-terminusof galanin and that the Trp residue in the corresponding positionin hGALR2 is involved in receptor subtype specificity of binding.Our model supports previous reports of Phe282 of hGALR1 interactingwith Trp2 of galanin and His264 of hGALR1 interacting with Tyr9of galanin.     

Molecular modelling of xylose isomerase catalysis: the role of electrostatics and charge transfer to metals

The two main steps of the mechanism of xylose-xylulose conversioncatalysed by D-xylose isomerase, the ring opening of xyloseand the isomerization of the opened product by hydride transfer,were investigated by molecular mechanical and molecular orbitaltechniques. The activation energies calculated for these reactionsclearly showed that hydrogen transfer is the rate-determiningstep of the enzymatic isomerization and that Mg²⁺ ions activatewhereas Zn²⁺ ions inhibit the reaction, in agreement with theexperiments. The remarkable differences between the net chargesof these ions found by molecular orbital calculations and theinspection of the protein electrostatic potential around thereaction intermediates indicate that the main role of bivalentmetal ions should be the electrostatic stabilization of thesubstrate transition states. In order to propose a more detailedmechanism, an attempt was made to clarify the effects of nearbyresidues (e.g. His54, Asp57, Lysl83, Asp257) in the reaction.Different isomerization mechanisms, such as through an enediolintermediate, were examined and could be excluded, in additionto the charge-relay mechanism during the ring opening.     

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.     

Molecular modelling and site-directed mutagenesis of the active site of endothelin-converting enzyme

Mammalian endothelin-converting enzyme is a membrane-bound metalloprotease; its C-terminal domain contains sequence motifs characteristic of zinc metalloproteases. We examined residues expected from molecular modelling to be important for substrate binding using selectively mutated recombinant rat ECE-1alpha expressed in CHO cells. A conserved N-A-Ar-Ar (Ar = aromatic) motif is likely to be important for substrate binding. Mutating N550 to Gln or Y552 to Phe reduces Vmax/Km by 8- and 18-fold, respectively. The equivalent residue to Y553 in thermolysin binds the inhibitor through its NH group. Removing this putative interaction by mutating Tyr to Pro destroys activity, but mutating it to Ala or Phe also removes most activity. Mutating G583 (in a conserved GGI motif N-terminal of the zinc-binding helix) to Ala has no measurable effect, but mutating G584 to Ala destroys activity. Changing V583 in the zinc-binding helix to Met, to mimic the sequence pattern in bovine ECE-2, increases Vmax/Km to 1.7-fold that of the wild- type. Assays of phosphoramidon binding follow the pattern of those of substrate binding, but the IC50 of the more potent ECE inhibitor CGS 26303 was not significantly altered by any of these mutations, suggesting that this compound may bind to ECE in a different mode from phosphoramidon.      

Structure and function of an antifreeze polypeptide from ocean pout, Macrozoarces americanus: role of glutamic acid residues in protein stability and antifreeze activity by site-directed mutagenesis

The successful expression and purification of the recombinantocean pout antifreeze polypeptide (rAFP) in Escherichia colihave enabled the study of its structure-function relationshipby site-directed mutagenesis. The role of carboxyl groups atGlu23 and Glu36 of the rAFP was probed by replacing these residueswith either glutamine or alanine residues as both single anddouble mutants. The AFP mutants were expressed, purified andcharacterized in terms of primary and secondary structures,thermal stability and antifreeze activities. The propertiesof these mutants were compared with those of the rAFP. Threedistinct functions are identified for the carboxyl groups: (i)the negative charges at positions 23 and 36 are involved inthe thermal stability of the polypeptide; (ii) the negativecharges at positions 23 and 36 contribute to the thermal hystereticactivities of the polypeptide; and (iii) the negative chargeat position 23 and hydrogen-bonding ability at position 36 contributeto the ice-binding activity of the polypeptide.     

Alteration of catalytic properties of chymosin by site-directed mutagenesis

Artificial mutations of chymosin by recombinant DNA techniqueswere generated to analyze the structure–function relationshipin this characteristic aspartk proteinase. In order to preparethe mutant enzymes in their active form, we established proceduresfor purification of correctly refolded prochymosin from inclusionbodies produced in Escherichia coli transformants and for itssubsequent activation. Mutagenesis by linker insertion intocDNA produced several mutants with an altered ratio of milkclotting activity to proteolytic activity and a different extentof stability. In addition to these mutants, several mutantswith a single amino acid exchange were also constructed by site-directedmutagenesis and kinetic parameters of these mutant enzymes weredetermined by using synthetic hexa- and octa-peptides as substrates.Exchange of Tyr75 on the flap of the enzyme to Phe caused amarked change of substrate specificity due to the change ofk_cat or K_m, depending on the substrate used. Exchange of Val110and Phe111 also caused a change of kinetic parameters, whichindicates functional involvement of these hydrophobic residuesin both the catalytic function and substrate binding. The mutantLys220–Leu showed a marked shift of the optimum pH tothe acidic side for hydrolysis of acid-denatured haemoglobinalong with a distinct increase in k_cat for the octa-peptidein a wide pH range.     

Directed evolution of Thermotoga neapolitana xylose isomerase: high activity on glucose at low temperature and low pH

The Thermotoga neapolitana xylose isomerase (TNXI) is extremelythermostable and optimally active at 95°C. Its derivative,TNXI Val185Thr (V185T), is the most active type II xylose isomerasereported, with a catalytic efficiency of 25.1 s^–1 mM^–1toward glucose at 80°C (pH 7.0). To further optimize TNXI’spotential industrial utility, two rounds of random mutagenesisand low temperature/low pH activity screening were performedusing the TNXI V185T-encoding gene as the template. Two highlyactive mutants were obtained, 3A2 (V185T/L282P) and 1F1 (V185T/L282P/F186S).1F1 was more active than 3A2, which in turn was more activethan TNXI V185T at all temperatures and pH values tested. 3A2and 1F1’s high activities at low temperatures were dueto significantly lower activation energies (57 and 44 kJ/mol,respectively) than that of TNXI and V185T (87 kJ/mol). MutationL282P introduced a kink in helix     

Improvement of thermal stability of Streptomyces cholesterol oxidase by random mutagenesis and a structural interpretation

Random mutagenesis was used to enhance the thermal stability of Streptomyces cholesterol oxidase. Four thermostable mutants were isolated and the following amino acid substitutions were identified: Ser103 to Thr (mutant S103T), Val121 to Ala (mutant V121A), Arg135 to His (mutant R135H) and Val145 to Glu (mutant V145E). The wild-type and mutant enzymes were purified and characterized. The properties of mutants S103T, V121A and R135H were similar to those of the wild type but they showed improved thermal stability. When the V145E mutation was introduced, the thermal stability of the enzyme was markedly increased and the optimum pH was desirably changed to encompass a broad range from acid to alkali. Analysis of multiple mutants constructed by site- directed mutagenesis showed that all the mutations except that of R135H had an additive influence on the other mutations. These mutational effects are discussed in terms of a three-dimensional structural model of the enzyme constructed on the basis of homology modelling.      

Study of B72.3 combining sites by molecular modeling and site-directed mutagenesis

A B72.3 Fab/sTn² complex was modeled from the known structureof B72.3 Fab and the dimeric Tn-serine cluster (sTn²). In thecomplex model, the side chains of 15 heavy- and light-chaincomplementarity-determining region (CDR) residues and the mainchains of two light-chain CDR residues contact the sTn² epitope.Among 15 CDR residues which contact sTn² in the model, two heavy-chainresidues (Ser95 and Tyr97) and light-chain CDR residue (Tyr96)have been confirmed in a previous study. To test the accuracyof the computational model, further site-directed mutagenesiswas performed by alanine scanning on the remaining 12 residuesthat are predicted in the model to have side-chain interactionswith sTn². Of these 12 mutants, eight that are all from theheavy-chain (His32Ala, Ala33Leu, Tyr50Ala, Ser52Ala, Asn52Ala,Asp56Ala, Lys58Ala and Tyr96Ala) had significantly reduced sTn²affinities, and four consisting of three light-chain mutations(Asn32Ala, Trp92Ala and Thr94Ala) and one heavy-chain mutation(His35Ala) retained wild-type sTn² affinity. On the whole, thisevidence suggests that the complex model, although not perfect,is correct in many of its features. In a more general vein,these results lend credibility to the computational modelingapproach for the study of the molecular basis of antigen–antibodycomplexes.     

Measurement of oxidation stability of polyolefins by thermal analysis

Differential thermal analysis (DTA) is well known for measuring temperatures and heats of physical and chemical transitions in materials. Recent development work on improved stabilization systems for polyolefin wire insulations has required fast procedures for measuring the performance of stabilization systems. Isothermal DTA procedures have been developed into routine tools for measuring stability of polyolefins by determining the oxidative induction time at elevated temperatures. Oxidative induction time has been used to study the stability of polyethylene and polypropylene in raw material form and on wire. The dependence on temperature and antioxidant concentration have been measured. Effects of oxidation catalysts, catalyst deactivators and various environmental exposures are readily observed. Techniques and apparatus have been developed for simultaneous testing of a multiplicity of samples, providing for economical large scale use of the method.     

The interpretation of site-directed mutagenesis experiments by linear free energy relations

Fersht and co-workers have applied a linear free energy relation(Brønsted equation) to analyze site-directed mutagenesisexperiments involving the enzyme tyrosyl-tRNA synthetase andhave suggested that the Brønsted exponent is linearlycorrelated with the value of the reaction coordinate at thetransition state. We point out that when the mutants differsolely through the formation or deletion of a hydrogen bondaway from the reaction center, a linear free energy relationis expected only in limiting cases for which the Brønstedrelation exponent is 0, 1 or . The results may be correlatedwith a conformational coordinate but not with the developmentof the reaction coordinate per se.     

Evolutionary trace analysis of TGF-{beta} and related growth factors: implications for site-directed mutagenesis

The TGF-ß family of growth factors contains a largenumber of homologous proteins, grouped in several subfamilieson the basis of sequence identity. These subgroups can be combinedinto three broader groups of related cytokines, with markedspecificities for their cellular receptors: the TGF-ßs,the activins and the BMPs/GDFs. Although structural informationis available for some members of the TGF-ß family,very little is known about the way in which these growth factorsinteract with the extra-cellular domains of their multiple cellsurface receptors or with the specific protein inhibitors thoughtto modulate their activity. In this paper, we use the evolutionarytrace method [Lichtarge et al. (1996) J. Mol. Biol., 257, 342–358]to locate two functional patches on the surface of TGF-ß-likegrowth factors. The first of these is centred on a conservedproline (P₃₆ in TGF-ßs 1–3) and contains twoamino acids which could account for the receptor specificityof TGF-ßs (H₃₄ and E₃₅). The second patch is locatedon the other side of the growth factor protomer and surroundsa hydrophobic cavity, large enough to accommodate the side chainof an aromatic residue. In addition to two conserved tryptophansat positions 30 and 32, the main protagonists in this potentialbinding interface are found at positions 31, 92, 93 and 98.Several mutagenesis studies have highlighted the importanceof the C-terminal region of the growth factor molecule in TGF-ßsand of residues in activin A equivalent to positions 31 and94 of the TGF-ßs for the binding of type II receptorsto these ligands. These data, together with our improved knowledgeof possible functional residues, can be used in future structure–functionanalysis experiments.     

Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis

Benzoylformate decarboxylase (BFD) from Pseudomonas putida wassubjected to directed molecular evolution to generate mutantswith increased carboligase activity which is a side reactionof the enzyme. After a single round of random mutagenesis mutantswere isolated which exhibited a 5-fold increased carboligaseactivity in aqueous buffer compared to the wild-type enzymewith a high enantiomeric excess of the product (S)-2-hydroxy-1-phenyl-propanone.From the same library, mutants with enhanced carboligase activityin water-miscible organic solvents have been isolated. The selectedmutants have been characterized by sequencing, revealing thatall mutants carry a mutation at Leu476, which is close to theactive site but does not directly interact with the active center.BFD-L476Q has a 5-fold higher carboligase activity than thewild-type enzyme. L476 was subjected to saturation mutagenesisyielding eight different mutants with up to 5-fold increasedcarboligase activity. Surprisingly, all L476 mutants catalyzethe formation of 2-hydroxy-1-phenyl-propanone with significantlyhigher enantioselectivity than the wild-type enzyme althoughenantioselectivity was not a selection parameter. Leu476 potentiallyplays the role of a gatekeeper of the active site of BFD, possiblyby controlling the release of the product. The biocatalyst couldbe significantly improved for its side reaction, the C–Cbond formation and for application under conditions that arenot optimized in nature.     

Improving activity and stability of cutinase towards the anionic detergent AOT by complete saturation mutagenesis

Cutinase is an enzyme suitable for detergent applications as well as for organic synthesis in non-aqueous solvents. However, its inactivation in the presence of anionic surfactants is a problem which we have addressed by creating a complete saturation library. For this, the cutinase gene from Fusarium solani pisi was mutated to incorporate all 19 possible amino acid exchanges at each of the 214 amino acid positions. The resulting library was screened for active variants with improved stability in the presence of the anionic surfactant dioctyl sulfosuccinate sodium salt (AOT). Twenty-four sites in cutinase were discovered where amino acid replacements resulted in a 2-11-fold stability increase as compared to the wild-type enzyme.     

Ni-olivine catalysts prepared by thermal impregnation: Structure, steam reforming activity, and stability

Ni-olivine catalysts prepared by thermal impregnation are candidate materials for in-bed catalytic tar removal during fluidized bed biomass gasification. The present work examined the structure and surface properties of the Ni-olivine catalysts prepared by thermal impregnation following preparation, during reduction by in-situ techniques, and following naphthalene-steam reforming in simulated biomass derived syngas. Catalysts were characterized by BET surface area, laser Raman spectroscopy (LRS), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). During the preparation, secondary phases containing Mg (i.e., (Mg,Fe)₃Si₂O₅(OH)₄ and MgFe₂O₄) reacted to form an olivine phase enriched in Mg and either MgO or Mg-enriched (Mg,Ni)O. The presence of excess steam in the naphthalene-steam reforming appeared to limit coking and also caused behavior to be different than was observed under reducing conditions. Post-reaction characterization indicated the loss of a metallic phase through a possible alloying process and the exchange of Fe for Mg in the olivine structure. Either can potentially explain the high coke resistance of the Ni-olivine catalysts prepared by thermal impregnation compared to Ni-olivine catalysts prepared by incipient wetness impregnation or the olivine support. Finally, the properties of the olivine catalysts with and without Ni were compared through activity for methanol- and ethylene-steam reforming.