The active site topology of Aspergillus niger endopolygalacturonase II as studied by site-directed mutagenesis

Strictly conserved charged residues among polygalacturonases (Asp-180, Asp-201, Asp-202, His-223, Arg-256, and Lys-258) were subjected to site-directed mutagenesis in Aspergillus niger endopolygalacturonase II. Specific activity, product progression, and kinetic parameters (K(m) and V(max)) were determined on polygalacturonic acid for the purified mutated enzymes, and bond cleavage frequencies on oligogalacturonates were calculated. Depending on their specific activity, the mutated endopolygalacturonases II were grouped into three classes. The mutant enzymes displayed bond cleavage frequencies on penta- and/or hexagalacturonate different from the wild type endopolygalacturonase II. Based on the biochemical characterization of endopolygalacturonase II mutants together with the three-dimensional structure of the wild type enzyme, we suggest that the mutated residues are involved in either primarily substrate binding (Arg-256 and Lys-258) or maintaining the proper ionization state of a catalytic residue (His-223). The individual roles of Asp-180, Asp-201, and Asp-202 in catalysis are discussed. The active site topology is different from the one commonly found in inverting glycosyl hydrolases.     

Catalytic and regulatory sites of yeast plasma membrane H(+)-ATPase studied by directed mutagenesis

More than 35 site-directed mutants of the plasma membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae have been constructed and expressed to investigate the function of N- and C-termini and of conserved amino acids. Conserved motif TGES seems to form part of both the catalytic machinery for the hydrolysis of the phosphorylated intermediate and the vanadate binding site. In addition, it is involved in the coupling of ATP hydrolysis to H+ transport. The phosphorylated intermediate is also essential for this coupling, but not for ATP hydrolysis. The aspartate residues of conserved motifs DPPR, TGD and TGDGVND (the last one) seem to form part of the ATP binding site. The positive charge of the conserved motif KGAP is important for the kinase or phosphorylating activity. A conserved proline and a conserved aspartate predicted to have a transmembrane location are essential for activity. The N-terminus contains a conserved acidic region which may be involved in assembly into the plasma membrane. All the hydrophobic stretches at the C-terminus are also required for assembly. The last 11 amino acids constitute a non-essential inhibitory domain involved in regulation of the enzyme by glucose metabolism.     

The ATP binding site of the yeast plasma membrane proton-translocating ATPase

Photoaffinity labeling of the active site of the yeast plasma membrane H(+)-ATPase has been studied with 2-azido-AMP and 2-azido-ATP. The ATPase activity of the enzyme decreases as the time of photolysis of the photoactive nucleotides in the presence of the enzyme increases. The covalent incorporation of [alpha-32P]2-azido-AMP into the enzyme and the inhibition of ATPase activity have comparable time courses. ATP protects the ATPase from incorporation of and photoinactivation by 2-azido-ATP or 2-azido-AMP. In the dark, 2-azido-ATP inhibits the ATPase at concentrations comparable to the apparent Michaelis constant for MgATP. After photolysis and proteolysis of the protein, three overlapping peptides labeled by the nucleotide analogues were purified by reversed-phase high performance liquid chromatography and sequenced. The peptides are derived from a region of the ATPase that is highly conserved in related cation pumps forming a phosphorylated intermediate during the catalytic cycle. Labeling with both nucleotide analogues occurs in peptides containing residues from aspartate 560 to lysine 566. The amino acids in this region conform to a consensus sequence for ATP binding derived from phosphofructokinase.     

Mapping of the active site of glutamate carboxypeptidase II by site-directed mutagenesis

Human glutamate carboxypeptidase II [GCPII (EC 3.4.17.21)] is recognized as a promising pharmacological target for the treatment and imaging of various pathologies, including neurological disorders and prostate cancer. Recently reported crystal structures of GCPII provide structural insight into the organization of the substrate binding cavity and highlight residues implicated in substrate/inhibitor binding in the S1' site of the enzyme. To complement and extend the structural studies, we constructed a model of GCPII in complex with its substrate, N-acetyl-l-aspartyl-l-glutamate, which enabled us to predict additional amino acid residues interacting with the bound substrate, and used site-directed mutagenesis to assess the contribution of individual residues for substrate/inhibitor binding and enzymatic activity of GCPII. We prepared and characterized 12 GCPII mutants targeting the amino acids in the vicinity of substrate/inhibitor binding pockets. The experimental results, together with the molecular modeling, suggest that the amino acid residues delineating the S1' pocket of the enzyme (namely Arg210) contribute primarily to the high affinity binding of GCPII substrates/inhibitors, whereas the residues forming the S1 pocket might be more important for the 'fine-tuning' of GCPII substrate specificity.     

Identification of the enzymatic active site of CD38 by site-directed mutagenesis

CD38 is a ubiquitous protein originally identified as a lymphocyte antigen and recently also found to be a multifunctional enzyme participating in the synthesis and metabolism of two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate. It is homologous to Aplysia ADP-ribosyl cyclase, where the crystal structure has been determined. Residues of CD38 corresponding to those at the active site of the Aplysia cyclase were mutagenized. Changing Glu-226, which corresponded to the catalytic residue of the cyclase, to Asp, Asn, Gln, Leu, or Gly eliminated essentially all enzymatic activities of CD38, indicating it is most likely the catalytic residue. Photoaffinity labeling showed that E226G, nevertheless, retained substantial NAD binding activity. The secondary structures of these inactive mutants as measured by circular dichroism were essentially unperturbed as compared with the wild type. Other nearby residues were also investigated. The mutants D147V and E146L showed 7- and 19-fold reduction in NADase activity, respectively. The cADPR hydrolase activity of the two mutants was similarly reduced. Asp-155, on the other hand, was crucial for the GDP-ribosyl cyclase activity since its substitution with either Glu, Asn, or Gln stimulated the activity 3-15-fold, whereas other activities remained essentially unchanged. In addition to these acidic residues, two tryptophans were also important, since all enzyme activities of W125F, W125Y, W189G and W189Y were substantially reduced. This is consistent with the two tryptophans serving a substrate positioning function. A good correlation was observed when the NADase activity of all the mutants was plotted against the cADPR hydrolase activity. Homology modeling revealed all these critical residues are clustered in a pocket near the center of the CD38 molecule. The results indicate a strong structural homology between the active sites of CD38 and the Aplysia cyclase.     

The putative effector-binding site of Leishmania mexicana pyruvate kinase studied by site-directed mutagenesis

The activity of pyruvate kinase of Leishmania mexicana is allosterically regulated by fructose 2,6-bisphosphate (F-2,6-P(2)), contrary to the pyruvate kinases from other eukaryotes that are usually stimulated by fructose 1,6-bisphosphate (F-1,6-P(2)). Based on the comparison of the three-dimensional structure of Saccharomyces cerevisiae pyruvate kinase crystallized with F-1,6-P(2) present at the effector site (R-state) and the L. mexicana enzyme crystallized in the T-state, two residues (Lys453 and His480) were proposed to bind the 2-phospho group of the effector. This hypothesis was tested by site-directed mutagenesis. The allosteric activation by F-2,6-P(2) appeared to be entirely abrogated in the mutated enzymes confirming our predictions.     

Water channel activity of radish plasma membrane aquaporins heterologously expressed in yeast and their modification by site-directed mutagenesis

Plants contain a number of aquaporin isoforms. We developed a method for determining the water channel activity of individual isoforms of aquaporin. Six plasma membrane aquaporins (RsPIPs) and two vacuolar membrane aquaporins (RsTIPs) of radish (Raphanus sativus) were expressed heterologously in Saccharomyces cerevisiae BJ5458, which is deficient in endogenous functional aquaporin. Aquaporins were detected by immunoblot analysis with corresponding antibodies. Water permeability of membranes from yeast transformants was assayed by stopped-flow spectrophotometry. The water channel activity of members of the RsPIP2 and RsTIP subfamilies was about 10 times and 5 times greater, respectively, than that of the control; however, RsPIP1s had little (RsPIP1-2 and RsPIP1-3) or no activity (RsPIP1-1). Site-directed mutation of several residues conserved in RsPIP1s or RsPIP2s markedly altered the water transport activity. Exchange of Ile244 of RsPIP1-3 with valine increased the activity to 250% of the wild type RsPIP1-3. On the other hand, exchange of Val235 of RsPIP2-2, which corresponds to RsPIP1-3 Ile244, with isoleucine caused a marked inactivation to 45% of the original RsPIP2-2. Mutation at possible phosphorylation sites at the N- and C-terminal tails also altered the activity. These results suggest that these residues in the half-helix loop E and the tails are involved in the water transport and the functional regulation of RsPIP1 and RsPIP2.     

Identification of active site residues of Escherichia coli fumarate reductase by site-directed mutagenesis.

Menaquinol-fumarate oxidoreductase of Escherichia coli is a four-subunit membrane-bound complex that catalyzes the final step in anaerobic respiration when fumarate is the terminal electron acceptor. The enzyme is structurally and catalytically similar to succinate dehydrogenase (succinate-ubiquinone oxidoreductase) from both procaryotes and eucaryotes. Both enzymes have been proposed to contain an essential cysteine residue at the active site based on studies with thiol-specific reagents. Chemical modification studies have also suggested roles for essential histidine and arginine residues in catalysis by succinate dehydrogenase. In the present study, a combination of site-directed mutagenesis and chemical modification techniques have been used to investigate the role(s) of the conserved histidine 232, cysteine 247, and arginine 248 residues of the flavorprotein subunit (FrdA) in active site function. A role for His-232 and Arg-248 of FrdA is shown by loss of both fumarate reductase and succino-oxidase activities following site-directed substitution of these particular amino acids. Evidence is also presented that suggests a second arginine residue may form part of the active site. Potential catalytic and substrate-binding roles for arginine are discussed. The effects of removing histidine-232 of FrdA are consistent with its proposed role as a general acid-base catalyst. The fact that succinate oxidation but not fumarate reduction was completely lost, however, might suggest that alternate proton donors substitute for His-232. The data confirm that cysteine 247 of FrdA is responsible for the N-ethylmaleimide sensitivity shown by fumarate reductase but is not required for catalytic activity or the tight-binding of oxalacetate, as previously thought.     

Evaluation by site-directed mutagenesis of active site amino acid residues of Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase

Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate, and carbon dioxide, and uses Mn²⁺ as the activating metal ion. The enzyme is a monomer and presents 68% identity with Escherichia coli PEP carboxykinase. Comparison with the crystalline structure of homologous E. coli PEP carboxykinase [Tari, L. W., Matte, A., Goldie, H., and Delbaere, L. T. J. (1997). Nature Struct. Biol. 4, 990–994] suggests that His225, Asp262, Asp263, and Thr249 are located in the active site of the protein, interacting with manganese ions. In this work, these residues were individually changed to Gln (His225) or Asn. The mutated enzymes present 3–6 orders of magnitude lower values of V _max/K _m, indicating high catalytic relevance for these residues. The His225Gln mutant showed increased K _m values for Mn²⁺ and PEP as compared with wild-type enzyme, suggesting a role of His²²⁵ in Mn²⁺ and PEP binding. From 1.5–1.6 Kcal/mol lower affinity for the 3(2)-O-(N-methylantraniloyl) derivative of adenosine diphosphate was observed for the His225Gln and Asp263Asn mutant A. succiniciproducens PEP carboxykinases, implying a role of His²²⁵ and Asp²⁶³ in nucleotide binding.     

Functional analysis of active site residues of Bacillus thuringiensis WB7 chitinase by site-directed mutagenesis

To investigate the roles of the active site residues in the catalysis of Bacillus thuringiensis WB7 chitinase, twelve mutants, F201L, F201Y, G203A, G203D, D205E, D205N, D207E, D207N, W208C, W208R, E209D and E209Q were constructed by site-directed mutagenesis. The results showed that the mutants F201L, G203D, D205N, D207E, D207N, W208C and E209D were devoid of activity, and the loss of the enzymatic activities for F201Y, G203A, D205E, W208R and E209Q were 72, 70, 48, 31 and 29%, respectively. The pH-activity profiles indicated that the optimum pH for the mutants as well as for the wildtype enzyme was 8.0. E209Q exhibited a broader active pH range while D205E, G203A and F201Y resulted in a narrower active pH range. The pH range of activity reduced 1 unit for D205E, and 2 units for G203A and F201Y. The temperature-activity profiles showed that the optimum temperature for other mutants as well as wildtype enzyme was 60°C, but 50°C for G203A, which suggested that G203A resulted in a reduction of thermostability. The study indicated that the six active site residues involving in mutagenesis played an important part in WB7 chitinase. In addition, the catalytic mechanisms of the six active site residues in WB7 chitinase were discussed.     

Identification of the enzymatic active site of tobacco caffeoyl-coenzyme A O-methyltransferase by site-directed mutagenesis.

Animal catechol O-methyltransferases and plant caffeoyl-coenzyme A O-methyltransferases share about 20% sequence identity and display common structural features. The crystallographic structure of rat liver catechol O-methyltransferase was used as a template to construct a homology model for tobacco caffeoyl-coenzyme A O-methyltransferase. Integrating substrate specificity data, the three-dimensional model identified several amino acid residues putatively involved in substrate binding. These residues were mutated by a polymerase chain reaction method and wild-type and mutant enzymes were each expressed in Escherichia coli and purified. Substitution of Arg-220 with Thr resulted in the total loss of enzyme activity, thus indicating that Arg-220 is involved in the electrostatic interaction with the coenzyme A moiety of the substrate. Changes of Asp-58 to Ala and Gln-61 to Ser were shown to increase K(m) values for caffeoyl coenzyme A and to decrease catalytic activity. Deletions of two amino acid sequences specific for plant enzymes abolished activity. The secondary structures of the mutants, as measured by circular dichroism, were essentially unperturbed as compared with the wild type. Similar changes in circular dichroism spectra were observed after addition of caffeoyl coenzyme A to the wild-type enzyme and the substitution mutants but not in the case of deletion mutants, thus revealing the importance of these sequences in substrate-enzyme interactions.     

Topology of the yeast plasma membrane proton-translocating ATPase

Four proteases have been used to assess the topology of the H+-ATPase from Saccharomyces cerevisiae reconstituted into phosphatidylserine vesicles. Limited proteolysis by trypsin and alpha-chymotrypsin inactivates the enzyme and produces stable, membrane-bound fragments. Sequence analyses of these peptides have located the peptide bonds hydrolyzed. The labile bonds are on opposite sides of a central hydrophilic domain containing consensus sequences for the site of phosphorylation and fluorescein isothiocyanate binding of several related ATPases. Limited proteolysis of the ATPase by elastase cuts approximately 50 amino acids from the C terminus, leaving the remaining membrane-bound fragments active. Proteolysis by carboxypeptidase Y in the presence and absence of detergent suggests that the C terminus is on the inside of the vesicle in this reconstitution. A model for the transmembrane arrangement of the polypeptide is proposed. In this model, the C terminus is on the inside of the vesicle, the N terminus is on the outside, the ATP binding region is on the outside, and the polypeptide passes through the membrane a minimum of five times.     

The study of the bacteriophage T5 deoxynucleoside monophosphate kinase active site by site-directed mutagenesis

The amino acid residues essential for the enzymatic activity of bacteriophage T5 deoxyribonucleoside monophosphate kinase were determined using a computer model of the enzyme active site. By site-directed mutagenesis, cloning, and gene expression in E. coli, a series of proteins were obtained with single substitutions of the conserved active site amino acid residues—S13A, D16N, T17N, T17S, R130K, K131E, Q134A, G137A, T138A, W150F, W150A, D170N, R172I, and E176Q. After purification by ion exchange and affine chromatography electrophoretically homogeneous preparations were obtained. The study of the enzymatic activity with natural acceptors of the phosphoryl group (dAMP, dCMP, dGMP, and dTMP) demonstrated that the substitutions of charged amino acid residues of the NMP binding domain (R130, R172, D170, and E176) caused nearly complete loss of enzymatic properties. It was found that the presence of the OH-group at position 17 was also important for the catalytic activity. On the basis of the analysis of specific activity variations we assumed that arginine residues at positions 130 and 172 were involved in the binding to the donor γ-phosphoryl and acceptor α-phosphoryl groups, as well as the aspartic acid residue at position 16 of the ATP-binding site (P-loop), in the binding to some acceptors, first of all dTMP. Disproportional changes in enzymatic activities of partially active mutants, G137A, T138A, T17N, Q134A, S13A, and D16N, toward different substrates may indicate that different amino acid residues participate in the binding to various substrates.     

Exploring the active site of yeast xylose reductase by site-directed mutagenesis of sequence motifs characteristic of two dehydrogenase/reductase family types

Starting from a common tyrosine, yeast xylose reductases (XRs) contain two conserved sequence motifs corresponding to the catalytic signatures of single-domain reductases/epimerases/dehydrogenases (Tyrⁿ-(X)₃-Lysⁿ⁺⁴) and aldo/keto reductases (AKRs) (Tyrⁿ-(X)₂₈-Lysⁿ⁺²⁹). Tyr⁵¹, Lys⁵⁵ and Lys⁸⁰ of XR from Candida tenuis were replaced by site-directed mutagenesis. The purified Tyr⁵¹→ Phe and Lys⁸⁰→Ala mutants showed turnover numbers and catalytic efficiencies for NADH-dependent reduction of -xylose between 2500- and 5000-fold below wild-type levels, suggesting a catalytic role of both residues. Replacing Lys⁵⁵ by Asn, a substitution found in other AKRs, did not detectably affect binding of coenzymes, and enzymatic catalysis to carbonyl/alcohol interconversion. The contribution of Tyr⁵¹ to rate enhancement of aldehyde reduction conforms with expectations for the general acid catalyst of the enzymatic reaction.     

Factors affecting the inhibition of yeast plasma membrane ATPase by vanadate

Inhibition of yeast plasma membrane ATPase by vanadate occurs only if either Mg2+ or MgATP2- is bound to the enzyme. The dissociation constant of the complex of vanadate and inhibitory sites is 0.14-0.20 microM in the presence of optimal concentrations of Mg2+ and of the order of 1 microM if the enzyme is saturated with MgATP2-. The dissociation constants of Mg2+ and MgATP2- for the sites involved are 0.4 and 0.62-0.73 mM, respectively, at pH 7. KCl does not increase the affinity of vanadate to the inhibitory sites as was found with (Na+ + K+)-ATPase. On the other hand, the effect of Mg2+ upon vanadate binding is similar to that upon (Na+ + K+)-ATPase, and the corresponding affinity constants of Mg2+ and vanadate for the two enzymes are of the same order of magnitude.     

A site-directed mutagenesis study of yeast calmodulin

A site-directed mutagenesis study was carried out in order to understand the regulatory mechanism of calmodulin. We started from the yeast (Saccharomyces cerevisiae) calmodulin gene since it has many differences in amino acid sequence and inferior functional properties compared with the vertebrate calmodulin. Recombinant yeast calmodulins were generated in Escherichia coli transformed by constructed expression plasmids. Three recombinant calmodulins were obtained. The first two were YCM61G, in which the Ca2(+)-binding site 2 (the four Ca2(+)-binding EF-hand structures in calmodulin were numbered from the N-terminus) was converted to the same as that in vertebrate calmodulin, and YCM delta 132-148, in which the C-terminal half sequence of site 4 was deleted. These two recombinant calmodulins had the same maximum Ca2+ binding (3 mol/mol) as yeast calmodulin, which indicates that site 4 of yeast calmodulin was the one losing Ca2+ binding capacity. YCM delta 132-148 could not activate target enzymes, whereas its Ca2+ binding profile was similar to those of yeast calmodulin and YCM61G. Therefore, the structure in site 4 which cannot bind Ca2+ is indispensable for the regulatory function of yeast calmodulin. The complete regulatory function of vertebrate calmodulin can be attained by the combination of 4 Ca2+ binding structures. The negative charge cluster in the central alpha-helix region is suggested to stabilize the active conformation of calmodulin, since the third yeast calmodulin mutant, YCM83E, which had the negative charge cluster, increased the maximum activation of myosin light chain kinase.     

Defining fibronectin's cell adhesion synergy site by site-directed mutagenesis

Fibronectin's RGD-mediated binding to the alpha5beta1 integrin is dramatically enhanced by a synergy site within fibronectin III domain 9 (FN9). Guided by the crystal structure of the cell-binding domain, we selected amino acids in FN9 that project in the same direction as the RGD, presumably toward the integrin, and mutated them to alanine. R1379 in the peptide PHSRN, and the nearby R1374 have been shown previously to be important for alpha5beta1-mediated adhesion (Aota, S., M. Nomizu, and K.M. Yamada. 1994. J. Biol. Chem. 269:24756-24761). Our more extensive set of mutants showed that R1379 is the key residue in the synergistic effect, but other residues contribute substantially. R1374A decreased adhesion slightly by itself, but the double mutant R1374A-R1379A was significantly less adhesive than R1379A alone. Single mutations of R1369A, R1371A, T1385A, and N1386A had negligible effects on cell adhesion, but combining these substitutions either with R1379A or each other gave a more dramatic reduction of cell adhesion. The triple mutant R1374A/P1376A/R1379A had no detectable adhesion activity. We conclude that, in addition to the R of the PHRSN peptide, other residues on the same face of FN9 are required for the full synergistic effect. The integrin-binding synergy site is a much more extensive surface than the small linear peptide sequence.     

Probing the mechanism of the bifunctional enzyme ketol-acid reductoisomerase by site-directed mutagenesis of the active site

Ketol-acid reductoisomerase (EC 1.1.1.86) is involved in the biosynthesis of the branched-chain amino acids. It is a bifunctional enzyme that catalyzes two quite different reactions at a common active site; an isomerization consisting of an alkyl migration, followed by an NADPH-dependent reduction of a 2-ketoacid. The 2-ketoacid formed by the alkyl migration is not released. Using the pure recombinant Escherichia coli enzyme, we show that the isomerization reaction has a highly unfavourable equilibrium constant. The reductase activity is shown to be relatively nonspecific and is capable of utilizing a variety of 2-ketoacids. The active site of the enzyme contains eight conserved polar amino acids and we have mutated each of these in order to dissect their contributions to the isomerase and reductase activities. Several mutations result in loss of the isomerase activity with retention of reductase activity. However, none of the 17 mutants examined have the isomerase activity only. We suggest a reason for this, involving direct reduction of a transition state formed during the isomerization, which is necessitated by the unfavourable equilibrium position of the isomerization. Our mechanism explains why the two activities must occur in a single active site without release of a 2-ketoacid and provides a rationale for the requirement for NADPH by the isomerase.     

Mapping the active site topography of the NAD-malic enzyme via alanine-scanning site-directed mutagenesis.

The NAD-malic enzyme cDNA has been subcloned into the pQE expression vector, expressed with a six-His tag, and purified. The His-tagged enzyme is purified by a combination of Ni-NTA and orange A agarose column chromatography with a yield of 45% and an estimated purity of >90%. The tag and linker have no effect on the kinetic parameters of the enzyme compared to the wild-type enzyme. Alanine-scanning site-directed mutagenesis has been carried out on all of the conserved neutral acid residues of the NAD-malic enzyme from Ascaris suum. Data obtained confirm the predicted role of D178 and D295 in metal ion binding, the likely role of D294, D361, and E440 in the NAD binding site, and the role of E58 and D272 in malate binding. Decreases in V/E(t) by 10(4)-fold and in V/K(malate)E(t) by 10(7)-fold, when D295 is changed to alanine, suggest that it is a likely candidate for the general base that accepts a proton from the malate hydroxyl in the oxidation step.     

Role of the amino acid invariants in the active site of MurG as evaluated by site-directed mutagenesis

To evaluate their role in the active site of the MurG enzyme from Escherichia coli, 13 residues conserved in the sequences of 73 MurG orthologues were submitted to site-directed mutagenesis. All these residues lay within, or close to, the active site of MurG as defined by its tridimensional structure [Ha et al., Prot. Sci. 9 (2000) 1045-1052, and Hu et al., Proc. Natl. Acad. Sci. USA 100 (2003) 845-849]. Thirteen mutants proteins, in which residues T15, H18, Y105, H124, E125, N127, N134, S191, N198, R260, E268, Q288 or N291 have been replaced by alanine, were obtained as the C-terminal His-tagged forms. The effects of the mutations on the activity were checked: (i) by functional complementation of an E. coli murG mutant strain by the mutated genes; and (ii) by the determination of the steady-state kinetic parameters of the purified proteins. Most mutations resulted in an important loss of activity and, in the case of N134A, in the production of a highly unstable protein. The results correlated with the assigned or putative functions of the residues based on the tridimensional structure.