Biomedical subjects

B W Matthews

Publications and source records attributed to B W Matthews.

At least 19 recordsLinked to original sources

PubMed1992

Structural comparison suggests that thermolysin and related neutral proteases undergo hinge-bending motion during catalysis.

Crystal structures are known for three members of the bacterial neutral protease family: thermolysin from Bacillus thermoproteolyticus (TLN), the neutral protease from Bacillus cereus (NEU), and the elastase of Pseudomonas aeruginosa (PAE), both in free and ligand-bound forms. Each enzyme consists of an N-terminal and C-terminal domain with the active site formed at the junction of the two domains. Comparison of the different molecules reveals that the structure within each domain is well conserved, but there are substantial hinge-bending displacements (up to 16 degrees) of one domain relative to the other. These domain motions can be correlated with the presence or absence of bound inhibitor, as was previously observed in the specific example of PAE [Thayer, M.M., Flaherty, K.M., & McKay, D.B. (1991) J. Biol. Chem. 266, 2864-2871]. The binding of inhibitor appears to be associated with a reduction of the domain hinge-bending angle by 6-14 degrees and a closure of the "jaws" of the active site cleft by about 2 A. Crystallographic refinement of the structure of thermolysin suggests that electron density seen in the active site of the enzyme in the original structure determination probably corresponds to a bound dipeptide. Thus, the crystal structure appears to correspond to an enzyme-inhibitor or enzyme-product complex, rather than the free enzyme, as has previously been assumed.

Amino Acid Sequence↗

PubMed1992

Structure of a hinge-bending bacteriophage T4 lysozyme mutant, Ile3-->Pro.

The mutant T4 phage lysozyme in which isoleucine 3 is replaced by proline (I3P) crystallizes in an orthorhombic form with two independent molecules in the asymmetric unit. Relative to wild-type lysozyme, which crystallizes in a trigonal form, the two I3P molecules undergo large hinge-bending displacements with the alignments of the amino-terminal and carboxy-terminal domains changed by 28.9 degrees and 32.9 degrees, respectively. The introduction of the mutation, together with the hinge-bending displacement, is associated with repacking of the side-chains of Phe4, Phe67 and Phe104. These aromatic residues are clustered close to the site of the mutation and are at the junction between the amino and carboxyl-terminal domains. As a result of this structural rearrangement the side-chain of Phe4 moves from a relatively solvent-exposed conformation to one that is largely buried. Mutant I3P also crystallizes in the same trigonal form as wild-type and, in this case, the observed structural changes are restricted to the immediate vicinity of the replacement. The main change is a shift of 0.3 to 0.5 A in the backbone of residues 1 to 5. The ability to crystallize I3P under similar conditions but in substantially different conformations suggests that the molecule undergoes large-scale hinge-bending displacements in solution. It is also likely that these conformational excursions are associated with repacking at the junction of the N-terminal and C-terminal domains. On the other hand, the analysis is complicated by possible effects of crystal packing. The different I3P crystal structures show substantial differences in the binding of solvent, both at the site of the Ile3-->Pro replacement and at other internal sites.

Bacteriophage T4↗

PubMed1992

Escherichia coli biotin holoenzyme synthetase/bio repressor crystal structure delineates the biotin- and DNA-binding domains.

The three-dimensional structure of BirA, the repressor of the Escherichia coli biotin biosynthetic operon, has been determined by x-ray crystallography and refined to a crystallographic residual of 19.0% at 2.3-A resolution. BirA is a sequence-specific DNA-binding protein that also catalyzes the formation of biotinyl-5'-adenylate from biotin and ATP and transfers the biotin moiety to other proteins. The level of biotin biosynthetic enzymes in the cell is controlled by the amount of biotinyl-5'-adenylate, which is the BirA corepressor. The structure provides an example of a transcription factor that is also an enzyme. The structure of BirA is highly asymmetric and consists of three domains. The N-terminal domain is mostly alpha-helical, contains a helix-turn-helix DNA-binding motif, and is loosely connected to the remainder of the molecule. The central domain consists of a seven-stranded mixed beta-sheet with alpha-helices covering one face. The other side of the sheet is largely solvent-exposed and contains the active site. The C-terminal domain comprises a six-stranded, antiparallel beta-sheet sandwich. The location of biotin binding is consistent with mutations that affect enzymatic activity. A nearby loop has a sequence that has been associated with phosphate binding in other proteins. It is inferred that ATP binds in this region, adjacent to the biotin. It is proposed that the binding of corepressor to monomeric BirA may promote DNA binding by facilitating the formation of a multimeric BirA-corepressor-DNA complex. The structural details of this complex remain an open question, however.

Adenosine Triphosphate↗

PubMed1992

Structural and thermodynamic analysis of compensating mutations within the core of chicken egg white lysozyme.

High resolution crystal structures have been determined for six chicken-type lysozymes that were constructed to investigate putative intermediates in the evolution of the lysozymes of modern game birds (Malcolm, B. A., Wilson, K. P., Matthews, B. W., Kirsch, J. F., and Wilson, A. C. (1990) Nature 345, 86-89). The amino acid replacements include Thr-40----Ser, Ile-55----Val, and Ser-91----Thr, as well as combinations of these substitutions. Residues 40, 55, and 91 are buried within the core of chicken lysozyme. The replacements therefore involve the insertion and/or removal of methyl groups from the protein interior. The mutant proteins have normal activities, and their thermal stabilities span a range of 7 degrees C, with some variants more stable and some less stable than the naturally occurring forms. Comparison of the crystal structures shows the overall structures to be very similar, but there are differences in the packing of side chains in the region of the replacements. The x-ray coordinates were used to evaluate the repacking of side chains in the protein interior and to attempt to evaluate the contributions of the different energetic interactions toward the overall stability of each variant. The results illustrate how proteins can compensate for potentially destabilizing substitutions in different ways and underscore the importance of high resolution structural data if changes in protein thermostability due to changes in protein sequence are to be understood. The findings also suggest that protein stability can be increased by mutations that lower strain in the protein interior while maintaining total buried hydrophobic surface area.

Egg White↗

PubMed1992

Folding and function of a T4 lysozyme containing 10 consecutive alanines illustrate the redundancy of information in an amino acid sequence.

Single and multiple Xaa----Ala substitutions were constructed in the alpha-helix comprising residues 39-50 in bacteriophage T4 lysozyme. The variant with alanines at 10 consecutive positions (A40-49) folds normally and has activity essentially the same as wild type, although it is less stable. The crystal structure of this polyalanine mutant displays no significant change in the main-chain atoms of the helix when compared with the wild-type structure. The individual substitutions of the solvent-exposed residues Asn-40, Ser-44, and Glu-45 with alanine tend to increase the thermostability of the protein, whereas replacements of the buried or partially buried residues Lys-43 and Leu-46 are destabilizing. The melting temperature of the lysozyme in which Lys-43 and Leu-46 are retained and positions 40, 44, 45, 47, and 48 are substituted with alanine (i.e., A40-42/44-45/47-49) is increased by 3.1 degrees C relative to wild type at pH 3.0, but reduced by 1.6 degrees C at pH 6.7. In the case of the charged amino acids Glu-45 and Lys-48, the changes in melting temperature indicate that the putative salt bridge between these two residues contributes essentially nothing to the stability of the protein. The results clearly demonstrate that there is considerable redundancy in the sequence information in the polypeptide chain; not every amino acid is essential for folding. Also, further evidence is provided that the replacement of fully solvent-exposed residues within alpha-helices with alanines may be a general way to increase protein stability. The general approach may permit a simplification of the protein folding problem by retaining only amino acids proven to be essential for folding and replacing the remainder with alanine.

Alanine↗

PubMed1992

Design and structural analysis of alternative hydrophobic core packing arrangements in bacteriophage T4 lysozyme.

An attempt has been made to design modified core-packing arrangements in bacteriophage T4 lysozyme. Alternative replacements of the buried residues Leu99, Met102, Val111 and Phe153 were selected using packing calculations and energy minimization. To test the design procedure, a series of multiple mutants was constructed culminating in the replacement L99F/M102L/V111I/F153L. These variants decrease the stability of T4 lysozyme by approximately 0 to 2 kcal/mol. The crystal structures of a number of the variants were determined. In the variant in which Val111 was replaced by Ile, alpha-helix 107-114 moved by approximately 1.5 A, breaking the hydrogen bond between the backbone carbonyl group of Thr109 and the backbone amide group of Gly113. This conformational change was not anticipated by the design procedure. Compensating interactions of magnitude up to 1.1 kcal/mol occur for some sets of mutations, while other sets display nearly additive stability changes. Within experimental error, the stability of the double mutant V111F/F153L is additive, with delta delta G different by only 0.1 kcal/mol from the sum of the two single mutants. The quadruple mutant L99F/M102L/V111I/F153L is destabilized by 0.5 kcal/mol, compared to delta delta G = -1.6 kcal/mol for the sum of the four single mutants. Multiple mutants show smaller overall structural changes from wild-type than M102L or V111I alone. Co-operative changes in structure and stability can be rationalized in terms of specific structural differences between single and multiple mutants. Genuine repacking of the hydrophobic core of T4 lysozyme with minimal effects on structure, stability and activity thus appears to have been achieved.

Computer Graphics↗

PubMed1992

Dissection of helix capping in T4 lysozyme by structural and thermodynamic analysis of six amino acid substitutions at Thr 59.

Threonine 59, a helix-capping residue at the amino terminus of the longest helix in T4 phage lysozyme, was substituted with valine, alanine, glycine, serine, asparagine, and aspartic acid. The valine, alanine, and glycine replacements were observed to be somewhat more destabilizing than serine, asparagine, and aspartic acid. The crystal structures of the different variants showed that changes in conformation occurred at the site of substitution, including Asp 61, which is nearby, as well as displacement of a solvent molecule that is hydrogen-bonded to the gamma-oxygen of Thr 59 in wild-type lysozyme. Neither the structures nor the stabilities of the mutant proteins support the hypothesis of Serrano and Fersht (1989) that glycine and alanine are better helix-capping residues than valine because a smaller-sized residue allows better hydration at the end of the helix. In the aspartic acid and asparagine replacements the substituted side chains form hydrogen bonds with the end of the helix, as does threonine and serine at this position. In contrast, however, the Asp and Asn side chains also make unusually close contacts with carbon atoms in Asp 61. This suggests a structural basis for the heretofore puzzling observations that asparagine is more frequently observed as a helix-capping residue than threonine [Richardson, J. S., & Richardson, D. C. (1988) Science 240, 1648-1652] yet Thr----Asn replacements at N-cap positions in barnase were found to be destabilizing [Serrano, L., & Fersht, A. R. (1989) Nature 342, 296-299].(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1992

Crystallization of beta-galactosidase from Escherichia coli.

Two crystal forms of beta-galactosidase have been obtained from Escherichia coli. One crystal form is hexagonal space group P6222 or enantiomorph, with cell dimensions a = b = 154 A, c = 750 A. The second form is monoclinic, space group P21, with cell dimensions a = 107.9 A, b = 207.5 A, c = 509.9 A, beta = 94.7 degrees. The monoclinic form seems better suited to detailed structural analysis. The crystals are radiation-sensitive, but by using synchrotron radiation in conjunction with a long (400 mm) crystal-to-film distance it was possible to resolve the individual reflections. On the basis of crystal density measurements, there are four tetramers each of molecular weight 465,000 per asymmetric unit. The Patterson function strongly suggests that two of the tetramers are related to the other two by translation. The data are consistent with the tetramers having 222 point symmetry, but this is not proven.

Crystallography↗

PubMed1992

Tolerance of T4 lysozyme to proline substitutions within the long interdomain alpha-helix illustrates the adaptability of proteins to potentially destabilizing lesions.

To investigate the ability of a protein to accommodate potentially destabilizing amino acid substitutions, and also to investigate the steric requirements for catalysis, proline was substituted at different sites within the long alpha-helix that connects the amino-terminal and carboxyl-terminal domains of T4 lysozyme. Of the four substitutions attempted, three yielded folded, functional proteins. The catalytic activities of these three mutant proteins (Q69P, D72P, and A74P) were 60-90% that of wild-type. Their melting temperatures were 7-12 degrees C less than that of wild-type at pH 6.5. Mutant D72P formed crystals isomorphous with wild-type allowing the structure to be determined at high resolution. In the crystal structure of wild-type lysozyme the interdomain alpha-helix has an overall bend angle of 8.5 degrees. In the mutant structure the introduction of the proline causes this bend angle to increase to 14 degrees and also causes a corresponding rotation of 5.5 degrees of carboxyl-terminal domain relative to the amino-terminal one. Except for the immediate location of the proline substitution there is very little change in the geometry of the interdomain alpha-helix. The results support the view that protein structures are adaptable and can compensate for potentially destabilizing amino acid substitutions. The results also suggest that the precise shape of the active site cleft of T4 lysozyme is not critical for catalysis.

Catalysis↗

PubMed1992

A cavity-containing mutant of T4 lysozyme is stabilized by buried benzene.

The hydrophobic cores of proteins are generally well packed, with few cavities. Mutations in which a bulky buried residue such as leucine or phenylalanine is replaced with a small residue such as alanine can create cavities in the core of a protein (our unpublished results). The sizes and shapes of such cavities can vary substantially depending on factors such as local geometry, whether or not a cavity already exists at the site of substitution, and the degree to which the protein structure relaxes to occupy the space vacated by the substituted residue. We show by crystallographic and thermodynamic analysis that the cavity created by the replacement Leu 99----Ala in T4 lysozyme is large enough to bind benzene and that ligand binding increases the melting temperature of the protein by 6.0 degrees C at pH 3.0. Benzene does not, however, bind to the cavity created by the Phe 153----Ala replacement. The results show that cavities can be engineered in proteins and suggest that such cavities might be tailored to bind specific ligands. The binding of benzene at an internal site 7 A from the molecular surface also illustrates the dynamic nature of proteins, even in crystals.

Amino Acid Sequence↗

PubMed1992

Response of a protein structure to cavity-creating mutations and its relation to the hydrophobic effect.

Six "cavity-creating" mutants, Leu46----Ala (L46A), L99A, L118A, L121A, L133A, and Phe153----Ala (F153A), were constructed within the hydrophobic core of phage T4 lysozyme. The substitutions decreased the stability of the protein at pH 3.0 by different amounts, ranging from 2.7 kilocalories per mole (kcal mol-1) for L46A and L121A to 5.0 kcal mol-1 for L99A. The double mutant L99A/F153A was also constructed and decreased in stability by 8.3 kcal mol-1. The x-ray structures of all of the variants were determined at high resolution. In every case, removal of the wild-type side chain allowed some of the surrounding atoms to move toward the vacated space but a cavity always remained, which ranged in volume from 24 cubic angstroms (A3) for L46A to 150 A3 for L99A. No solvent molecules were observed in any of these cavities. The destabilization of the mutant Leu----Ala proteins relative to wild type can be approximated by a constant term (approximately 2.0 kcal mol-1) plus a term that increases in proportion to the size of the cavity. The constant term is approximately equal to the transfer free energy of leucine relative to alanine as determined from partitioning between aqueous and organic solvents. The energy term that increases with the size of the cavity can be expressed either in terms of the cavity volume (24 to 33 cal mol-1 A-3) or in terms of the cavity surface area (20 cal mol-1 A-2). The results suggest how to reconcile a number of conflicting reports concerning the strength of the hydrophobic effect in proteins.

Amino Acid Sequence↗

PubMed1992

Analysis of the effectiveness of proline substitutions and glycine replacements in increasing the stability of phage T4 lysozyme.

It was previously shown that the two replacements Gly 77-->Ala (G77A) and Ala 82-->Pro (A82P) increase the thermostability of phage T4 lysozyme at pH 6.5. Such replacements are presumed to restrict the degrees of freedom of the unfolded protein and so decrease the entropy of unfolding [B. W. Matthews, H. Nicholson, and W. J. Becktel (1987) Proceedings of the National Academy of Science USA Vol. 84, pp. 6663-6667]. To further test this approach, three additional replacements--G113A, K60P and A93P--have been constructed. On the basis of model building, each of these three replacements was judged to be less than optimal because it would tend to introduce unfavorable van der Waals contacts with neighboring parts of the protein. The presence of such contacts was verified for G113A and K60P by conformational adjustments seen in the crystal structures of these mutant proteins. In the case of G113A there are backbone conformational changes of 0.5-1.0 A in the short alpha-helix, 108-113, that includes the site of substitution. In the case of K60P the pyrrolidine ring shows evidence of strain. The thermal stability of each of the three variants at both pH 2.0 and pH 6.5 was found to be very close to that of wild-type lysozyme. The results suggest that the procedure used to predict sites for both Xaa-->Pro and Gly-->Ala is, in principle, correct. At the same time, the increase in stability expected from substitutions of this type is modest, and can easily be offset by strain associated with introduction of the alanine or proline. This means that the criteria used to select substitutions that will increase thermostability have to be stringent at least. In the case of T4 lysozyme this severely limits the number of sites. The analysis reveals a significant discrepancy between the conformational energy surface predicted for the residue preceding a proline and the conformations observed in crystal structures.

Bacteriophage T4↗

PubMed1992

Flexible-geometry conformational energy maps for the amino acid residue preceding a proline.

Previously calculated conformational energy maps suggest that the alpha-helical conformation for the residue preceding a proline is disfavored relative to the extended conformation by more than 7 kcal/mol. In known protein structures this conformation is observed, however, to occur for about 9% of all prolines. In addition, introduction or removal of prolines at theoretically unfavorable positions in proteins and peptides can have modest effects on stability and structure. To investigate the discrepancy between calculation and experiment, we have determined how the conformation of the proline affects the calculated energy. We have also explored the effect of bond length and bond angle relaxation on the conformational energy map. The conformational energy of the preceding residue is found to be unaffected by the conformation of the proline, but the effect of allowing covalent bond relaxation is dramatic. If bond lengths and angles, and dihedral angles within the pyrrolidine ring, are allowed to relax, a calculated energy difference between the alpha and beta conformations of 1.1 kcal/mol is obtained, in reasonable agreement with experiment. The detailed shape of the calculated energy surface is also in excellent agreement with the observed conformational distributions in known protein structures.

Amino Acid Sequence↗

PubMed1992

Structure of a stabilizing disulfide bridge mutant that closes the active-site cleft of T4 lysozyme.

The engineered disulfide bridge between residues 21 and 142 of phage T4 lysozyme spans the active-site cleft and can be used as a switch to control the activity of the enzyme (Matsumura, M. & Matthews, B.W., 1989, Science 243, 792-794). In the oxidized form the disulfide increases the melting temperature of the protein by 11 degrees C at pH 2. The crystal structure of this mutant lysozyme has been determined in both the reduced and oxidized forms. In the reduced form, the crystal structure of the mutant is shown to be extremely similar to that of wild type. In the oxidized form, however, the formation of the disulfide bridge causes the alpha-carbons of Cys 21 and Cys 142, on opposite sides of the active-site cleft, to move toward each other by 2.5 A. In association with this movement, the amino-terminal domain of the protein undergoes a rigid-body rotation of 5.1 degrees relative to the carboxy-terminal domain. This rotation occurs about an axis passing through the junction of the amino-terminal and carboxy-terminal domains and is also close to the axis that best fits the apparent thermal motion of the amino-terminal domain seen previously in crystals of wild-type lysozyme. Even though the engineered Cys 21-Cys 142 disulfide links together the amino-terminal and carboxy-terminal domains of T4 lysozyme, it does not reduce the apparent mobility of the one domain relative to the other. The pronounced "hinge-bending" mobility of the amino-terminal domain that is suggested by the crystallographic thermal parameters of wild-type lysozyme persists in the oxidized (and reduced) mutant structures. In the immediate vicinity of the introduced disulfide bridge the mutant structure is more mobile (or disordered) than wild type, so much so that the exact conformation of Cys 21 remains obscure. As with the previously described disulfide bridge between residues 9 and 164 of T4 lysozyme (Pjura, P.E., Matsumura, M., Wozniak, J.A., & Matthews, B.W., 1990, Biochemistry 29, 2592-2598), the engineered cross-link substantially enhances the stability of the protein without making the folded structure more rigid.

Bacteriophage T4↗

PubMed1992

Multiple alanine replacements within alpha-helix 126-134 of T4 lysozyme have independent, additive effects on both structure and stability.

In a systematic attempt to identify residues important in the folding and stability of T4 lysozyme, five amino acids within alpha-helix 126-134 were substituted by alanine, either singly or in selected combinations. Together with three alanines already present in the wild-type structure this provided a set of mutant proteins with up to eight alanines in sequence. All the variants behaved normally, suggesting that the majority of residues in the alpha-helix are nonessential for the folding of T4 lysozyme. Of the five individual alanine substitutions it is inferred that four result in slightly increased protein stability and one, the replacement of a buried leucine with alanine, substantially decreased stability. The results support the idea that alanine is a residue of high helix propensity. The change in protein stability observed for each of the multiple mutants is approximately equal to the sum of the energies associated with each of the constituent substitutions. All of the variants could be crystallized isomorphously with wild-type lysozyme, and, with one trivial exception, their structures were determined at high resolution. Substitution of the largely solvent-exposed residues Asp 127, Glu 128, and Val 131 with alanine caused essentially no change in structure except at the immediate site of replacement. Substitutions of the partially buried Asn 132 and the buried Leu 133 with alanine were associated with modest (< or = 0.4 A) structural adjustments. The structural changes seen in the multiple mutants were essentially a combination of those seen in the constituent single replacements. The different replacements therefore act essentially independently not only so far as changes in energy are concerned but also in their effect on structure. The destabilizing replacement Leu 133-->Ala made alpha-helix 126-134 somewhat less regular. Incorporation of additional alanine replacements tended to make the helix more uniform. For the penta-alanine variant a distinct change occurred in a crystal-packing contact, and the "hinge-bending angle" between the amino- and carboxy-terminal domains changed by 3.6 degrees. This tends to confirm that such hinge-bending in T4 lysozyme is a low-energy conformational change.

Alanine↗

PubMed1992

Dissection of protein structure and folding by directed mutagenesis.

The lysozyme from bacteriophage T4 is being used as a model system to determine the roles of individual amino acids in the folding and stability of a typical globular protein. One general finding is that the protein is very adaptable, being able to accommodate many potentially destabilizing replacements. In order to determine the importance of 'alpha-helix propensity' in protein stability, different replacements have been made within alpha-helical segments of T4 lysozyme. Several such substitutions of the form Xaa-->Ala increase the stability of the protein, supporting the idea that alanine is a strongly helix-favouring amino acid. It is possible to engineer a protein that has up to ten alanines in succession, yet still folds and has normal activity. This illustrates the redundancy that is present in the amino acid sequence. A number of 'cavity-creating' mutants of the form Leu-->Ala have been constructed to understand better the nature of hydrophobic stabilization. The structural consequences of these mutations differ from site to site. In some cases the protein structure hardly changes at all; in other cases removal of the wild-type side-chain allows surrounding atoms to move in and occupy the vacated space, although a cavity always remains. The destabilization of the protein associated with these cavity-creating mutations also varies from case to case. The results suggest how to reconcile recent conflicting reports concerning the strength of the hydrophobic effect in proteins.

Bacteriophage T4↗

PubMed1992

Structural basis for the action of thermolysin.

High resolution X-ray crystallography has been used to determine the modes of binding to thermolysin of a series of different inhibitors including dipeptides, mercaptans, hydroxamates, N-carboxymethyl peptides and phosphonamidates. The interactions displayed by such inhibitors illustrate interactions that are presumed to occur between the enzyme and its substrates during catalysis. The crystallographic analysis, together with model building, suggest a detailed stereochemical mechanism of action for thermolysin and, by analogy, other zinc proteases such as carboxypeptidase A and the angiotensin converting enzyme. Analysis of a series of phosphonamidates, which are presumed to be transition-state analogues, has shown that chemically similar inhibitors can adopt dissimilar modes of binding. These different configurations provide a rationalization for large differences in the kinetics of binding that are observed for these inhibitors. Experiments with thermolysin as a test case suggest that knowledge of the three-dimensional structure of an enzyme or receptor will greatly facilitate the rational design of drugs directed at such targets.

Amino Acid Sequence↗

PubMed1991

Structural and thermodynamic consequences of burying a charged residue within the hydrophobic core of T4 lysozyme.

To determine the energetic and structural consequences of placing a charged group within the core of a protein, two "buried charge" mutants, Met 102----Lys (M102K) and Leu 133----Asp (L133D) were constructed in phage T4 lysozyme. Both proteins fold at neutral pH, although they are substantially less stable than wild type. The activity of M102K is about 35% that of wild type, while that of L133D is about 4%. M102K could be crystallized, and its structure was determined at high resolution. The crystal structure (at pH 6.8) of the mutant is very similar to that of wild type except for the alpha-helix that includes residues 108-113. In wild-type lysozyme, one side of this helix is exposed to solvent and the other contacts Met 102. In the M102K structure this alpha-helix becomes much more mobile, possibly allowing partial access of Lys 102 to solvent. The stability of M102K, determined by monitoring the unfolding of the protein with CD, is pH-dependent, consistent with the charged form of the substituted amino acid being more destabilizing than the uncharged form. The pKa of Lys 102 was estimated to be 6.5 both by differential titration and also by NMR analysis of isotopically labeled protein with 13C incorporated at the C epsilon position of all lysines. As the pH is lowered below pH 6.5, the overall three-dimensional structure of M102K at room temperature appears to be maintained to pH 3 or so, although there is evidence for some structural adjustment possibly allowing solvent accessibility to the protonated form of Lys 102.

Amino Acid Sequence↗