Analysis of a conserved hydrophobic pocket important for the thermostability of Bacillus pumilus chloramphenicol acetyltransferase (CAT-86)

Site-directed mutagenesis was carried out on Bacillus pumilus chloramphenicol acetyltransferase (CAT-86) to determine the effects of substitution at a conserved hydrophobic pocket identified earlier as important for thermostability. Mutations were introduced that would substitute residues at consensus positions 33, 191 and 203 in the enzyme, both individually and in combination. Two mutants, SDM1 (CAT-86 Y33F, A203V) and SDM5 (CAT-86 A203I), were more thermostable than wild-type and two mutants, SDM4 (CAT-86 I191V) and SDM7 (CAT-86 A203G), were less stable. Reconstruction of the residues of this hydrophobic pocket to that of a more thermostable CAT-R387 enzyme pocket (as a Y33F, I191V, A203V triple mutant) increased the thermostability of the enzyme above the wild-type, but its stability was less than that of SDM1 and SDM5. The K(m) values of the mutant enzymes for chloramphenicol and acetyl-CoA were essentially unaltered (in the ranges 15-30 and 26-35 microM respectively) and the specific activity of purified enzyme was in the range 270-710 units/mg protein. The possible effects of the amino acid substitutions on the CAT-86 structure were determined by homology modelling. A reduction in conformational strain and optimized hydrophobic interactions are predicted to be responsible for the increased thermostability of the SDM1 and SDM5 mutants.     

Application of Rigidity Theory to the Thermostabilization of Lipase A from Bacillus subtilis

Protein thermostability is a crucial factor for biotechnological enzyme applications. Protein engineering studies aimed at improving thermostability have successfully applied both directed evolution and rational design. However, for rational approaches, the major challenge remains the prediction of mutation sites and optimal amino acid substitutions. Recently, we showed that such mutation sites can be identified as structural weak spots by rigidity theory-based thermal unfolding simulations of proteins. Here, we describe and validate a unique, ensemble-based, yet highly efficient strategy to predict optimal amino acid substitutions at structural weak spots for improving a protein’s thermostability. For this, we exploit the fact that in the majority of cases an increased structural rigidity of the folded state has been found as the cause for thermostability. When applied prospectively to lipase A from Bacillus subtilis, we achieved both a high success rate (25% over all experimentally tested mutations, which raises to 60% if small-to-large residue mutations and mutations in the active site are excluded) in predicting significantly thermostabilized lipase variants and a remarkably large increase in those variants’ thermostability (up to 6.6°C) based on single amino acid mutations. When considering negative controls in addition and evaluating the performance of our approach as a binary classifier, the accuracy is 63% and increases to 83% if small-to-large residue mutations and mutations in the active site are excluded. The gain in precision (predictive value for increased thermostability) over random classification is 1.6-fold (2.4-fold). Furthermore, an increase in thermostability predicted by our approach significantly points to increased experimental thermostability (p < 0.05). These results suggest that our strategy is a valuable complement to existing methods for rational protein design aimed at improving thermostability.     

Increased thermostability of microbial transglutaminase by combination of several hot spots evolved by random and saturation mutagenesis

The thermostability of microbial transglutaminase (MTG) of Streptomyces mobaraensis was further improved by saturation mutagenesis and DNA-shuffling. High-throughput screening was used to identify clones with increased thermostability at 55°C. Saturation mutagenesis was performed at seven "hot spots", previously evolved by random mutagenesis. Mutations at four positions (2, 23, 269, and 294) led to higher thermostability. The variants with single amino acid exchanges comprising the highest thermostabilities were combined by DNA-shuffling. A library of 1,500 clones was screened and variants showing the highest ratio of activities after incubation for 30 min at 55°C relative to a control at 37°C were selected. 116 mutants of this library showed an increased thermostability and 2 clones per deep well plate were sequenced (35 clones). 13 clones showed only the desired sites without additional point mutations and eight variants were purified and characterized. The most thermostable mutant (triple mutant S23V-Y24N-K294L) exhibited a 12-fold higher half-life at 60°C and a 10-fold higher half-life at 50°C compared to the unmodified recombinant wild-type enzyme. From the characterization of different triple mutants differing only in one amino acid residue, it can be concluded that position 294 is especially important for thermostabilization. The simultaneous exchange of amino acids at sites 23, 24, 269 and 289 resulted in a MTG-variant with nearly twofold higher specific activity and a temperature optimum of 55°C. A triple mutant with amino acid substitutions at sites 2, 289 and 294 exhibits a temperature optimum of 60°C, which is 10°C higher than that of the wild-type enzyme.     

耐热荧光素酶的表达及热稳定焦磷酸测序体系的建立

焦磷酸测序是一种基于生物发光反应的实时DNA序列测定技术,其反应体系中的荧光素酶决定测序反应的灵敏度。传统焦测序反应使用的是野生型北美荧光素酶Photinus pyralis luciferase(PpL),该酶热稳定性较差,因此由该酶配制的焦磷酸测序反应体系对热不稳定,影响焦磷酸测序的灵敏度。为了建立热稳定的焦磷酸测序反应体系,全合成了耐热突变日本萤火虫荧光素酶的编码序列,并将其插入到表达载体pET28a(+)中构建重组质粒,将重组质粒转化到大肠杆菌中,筛选得到的阳性重组菌成功表达了N端带有6×His(组氨酸)标签的耐热突变日本萤火虫荧光素酶。利用镍亲和层析法,纯化得到了分子量约为60 kDa且比活为4.29×1010RLU/mg的重组蛋白。对重组耐热日本萤火虫荧光素酶的热稳定性研究结果表明,该酶在50℃时仍具有活性,在40℃下孵育25 min后活性保留了90%以上。与基于野生型北美荧光素酶的焦磷酸测序体系相比,由耐热日本萤火虫荧光素酶配制的测序反应体系对热稳定性有了极大的提高,该测序反应液在37℃放置1 h后仍能够得到正确的测序结果。研究结果为建立稳定可靠的现场及时快速焦磷酸测序反应体系奠定了基础。     

A Dynamic Hydrophobic Core and Surface Salt Bridges Thermostabilize a Designed Three-Helix Bundle

Thermostable proteins are advantageous in industrial applications, as pharmaceuticals or biosensors, and as templates for directed evolution. As protein-design methodologies improve, bioengineers are able to design proteins to perform a desired function. Although many rationally designed proteins end up being thermostable, how to intentionally design de novo, thermostable proteins is less clear. UVF is a de novo-designed protein based on the backbone structure of the Engrailed homeodomain (EnHD) and is highly thermostable (T_m > 99°C vs. 52°C for EnHD). Although most proteins generally have polar amino acids on their surfaces and hydrophobic amino acids buried in their cores, protein engineers followed this rule exactly when designing UVF. To investigate the contributions of the fully hydrophobic core versus the fully polar surface to UVF’s thermostability, we built two hybrid, chimeric proteins combining the sets of buried and surface residues from UVF and EnHD. Here, we determined a structural, dynamic, and thermodynamic explanation for UVF’s thermostability by performing 4 μs of all-atom, explicit-solvent molecular dynamics simulations at 25 and 100°C, Tanford-Kirkwood solvent accessibility Monte Carlo electrostatic calculations, and a thermodynamic analysis of 40 temperature runs by the weighted-histogram analysis method of heavy-atom, structure-based models of UVF, EnHD, and both chimeric proteins. Our models showed that UVF was highly dynamic because of its fully hydrophobic core, leading to a smaller loss of entropy upon folding. The charged residues on its surface made favorable electrostatic interactions that contributed enthalpically to its thermostability. In the chimeric proteins, both the hydrophobic core and charged surface independently imparted thermostability.     

Relative tolerance of mesostable and thermostable protein homologs to extensive mutation

Evolvability, designability, and plasticity of a protein are properties that are important to protein engineers, but difficult to quantify. Here, we directly compare homologous AroQ chorismate mutases from the thermophile Methanococcus jannaschii and the mesophile Escherichia coli with respect to their capacity to accommodate extensive mutation. The N-terminal helix comprising about 40% of these proteins was randomized at the genetic level using a binary pattern of hydrophobic and hydrophilic residues based on the respective wild-type sequences. Catalytically active library members were identified by a survival-selection assay in a chorismate mutase-deficient E. coli strain. Functional variants were found approximately approximately 10-times more frequently with the thermostable protein compared to its mesostable counterpart. Moreover, detailed sequence analysis revealed that functional M. jannaschii enzyme variants contained a smaller number of conserved residues and tolerated greater variability at individual sequence positions. Our results thus highlight the greater robustness of the thermostable protein with respect to amino acid substitution, while identifying specific sites important for constructing active enzymes. Overall, they support the notion that redesign projects will benefit from using a thermostable starting structure, even at very high mutational loads.     

Structural features of thermozymes

Enzymes synthesized by thermophiles and hyperthermophiles are known as thermozymes. These enzymes are typically thermostable, or resistant to irreversible inactivation at high temperatures, and thermophilic, i.e. optimally active at elevated temperatures between 60 and 125 degrees C. Enzyme thermostability encompasses thermodynamic stability and kinetic stability. Thermodynamic stability is defined by the enzyme's free energy of stabilization (deltaG(stab)) and by its melting temperature (Tm). An enzyme's kinetic stability is often expressed as its halflife (t1/2) at defined temperature. DeltaG(stab) of thermophilic proteins is 5-20 kcal/mol higher than that of mesophilic proteins. The thermostability mechanisms for thermozymes are varied and depend on the enzyme; nevertheless, some common features can be identified as contributing to stability. These features include more interactions (i.e. hydrogen bonds, electrostatic interactions, hydrophobic interactions, disulfide bonds, metal binding) than in less stable enzymes and superior conformational structure (i.e. more rigid, higher packing efficiency, reduced entropy of unfolding, conformational strain release and stability of alpha-helix). Understanding of the stabilizing features will greatly facilitate reengineering of some of the mesozymes to more stable thermozymes.     

Prediction of thermostability from amino acid attributes by combination of clustering with attribute weighting: a new vista in engineering enzymes

The engineering of thermostable enzymes is receiving increased attention. The paper, detergent, and biofuel industries, in particular, seek to use environmentally friendly enzymes instead of toxic chlorine chemicals. Enzymes typically function at temperatures below 60°C and denature if exposed to higher temperatures. In contrast, a small portion of enzymes can withstand higher temperatures as a result of various structural adaptations. Understanding the protein attributes that are involved in this adaptation is the first step toward engineering thermostable enzymes. We employed various supervised and unsupervised machine learning algorithms as well as attribute weighting approaches to find amino acid composition attributes that contribute to enzyme thermostability. Specifically, we compared two groups of enzymes: mesostable and thermostable enzymes. Furthermore, a combination of attribute weighting with supervised and unsupervised clustering algorithms was used for prediction and modelling of protein thermostability from amino acid composition properties. Mining a large number of protein sequences (2090) through a variety of machine learning algorithms, which were based on the analysis of more than 800 amino acid attributes, increased the accuracy of this study. Moreover, these models were successful in predicting thermostability from the primary structure of proteins. The results showed that expectation maximization clustering in combination with uncertainly and correlation attribute weighting algorithms can effectively (100%) classify thermostable and mesostable proteins. Seventy per cent of the weighting methods selected Gln content and frequency of hydrophilic residues as the most important protein attributes. On the dipeptide level, the frequency of Asn-Glu was the key factor in distinguishing mesostable from thermostable enzymes. This study demonstrates the feasibility of predicting thermostability irrespective of sequence similarity and will serve as a basis for engineering thermostable enzymes in the laboratory.     

Hyperthermostabilization of Bacillus licheniformis alpha-amylase and modulation of its stability over a 50 degrees C temperature range

Bacillus licheniformis alpha-amylase (BLA) is a highly thermostable starch-degrading enzyme that has been extensively studied in both academic and industrial laboratories. For over a decade, we have investigated BLA thermal properties and identified amino acid substitutions that significantly increase or decrease the thermostability. This paper describes the cumulative effect of some of the most beneficial point mutations identified in BLA. Remarkably, the Q264S-N265Y double mutation led to a rather limited gain in stability but significantly improved the amylolytic function. The most hyperthermostable variants combined seven amino acid substitutions and inactivated over 100 times more slowly and at temperatures up to 23 degrees C higher than the wild-type enzyme. In addition, two highly destabilizing mutations were introduced in the metal binding site and resulted in a decrease of 25 degrees C in the half-inactivation temperature of the double mutant enzyme compared with wild-type. These mutational effects were analysed by protein modelling based on the recently determined crystal structure of a hyperthermostable BLA variant. Our engineering work on BLA shows that the thermostability of an already naturally highly thermostable enzyme can be substantially improved and modulated over a temperature range of 50 degrees C through a few point mutations.     

Interdomain Hydrophobic Interactions Modulate the Thermostability of Microbial Esterases from the Hormone-Sensitive Lipase Family

Microbial hormone-sensitive lipases (HSLs) contain a CAP domain and a catalytic domain. However, it remains unclear how the CAP domain interacts with the catalytic domain to maintain the stability of microbial HSLs. Here, we isolated an HSL esterase, E40, from a marine sedimental metagenomic library. E40 exhibited the maximal activity at 45 °C and was quite thermolabile, with a half-life of only 2 min at 40 °C, which may be an adaptation of E40 to the permanently cold sediment environment. The structure of E40 was solved to study its thermolability. Structural analysis showed that E40 lacks the interdomain hydrophobic interactions between loop 1 of the CAP domain and α7 of the catalytic domain compared with its thermostable homologs. Mutational analysis showed that the introduction of hydrophobic residues Trp²⁰² and Phe²⁰³ in α7 significantly improved E40 stability and that a further introduction of hydrophobic residues in loop 1 made E40 more thermostable because of the formation of interdomain hydrophobic interactions. Altogether, the results indicate that the absence of interdomain hydrophobic interactions between loop 1 and α7 leads to the thermolability of E40. In addition, a comparative analysis of the structures of E40 and other thermolabile and thermostable HSLs suggests that the interdomain hydrophobic interactions between loop 1 and α7 are a key element for the thermostability of microbial HSLs. Therefore, this study not only illustrates the structural element leading to the thermolability of E40 but also reveals a structural determinant for HSL thermostability.     

Discrimination of thermostable and thermophilic lipases using support vector machines

Discriminating thermophilic lipases from their similar thermostable counterparts is a challenging task and it would help to design stable proteins. In this study, the distributions of N (N=2, 3) neighboring amino acids and the non-adjacent di-residue coupling patterns in the sequences of 65 thermostable and 77 thermophilic lipases had been systematically analyzed. It was found that the hydrophobic residues Leu, Pro, Met, Phe, Trp, as well as the polar residue Tyr had higher occurrence in thermophilic lipases than thermostable ones. The occurrence frequencies of KC EE KE RE, VE, YI, EK, VK, EV, YV, EY, KY, VY and YY in thermophilic proteins were significantly higher, while the occurrence frequencies of QC, QH, QN, HQ, MQ, NQ, QQ, TQ, QS and QT were significantly lower. CXP or CPX showed significantly positive to lipase thermostability, while XXQ or QXX showed significantly negative to lipase thermostability. Non-adjacent di-residue coupling patterns of PR14, RY32, YR47, LE53, LE64, PP64, RP70 and PP101 were significantly different in thermophilic lipases and their thermostable counterparts. The composition of dipeptide, tripeptide and non-adjacent di-residue patterns contained more information than amino acid composition. A statistical method based on support vector machines (SVMs) was developed for discriminating thermophilic and thermostable lipases. The accuracy of this method for the training dataset was 97.17?. Furthermore, the highest accuracy of the method for testing datasets was 98.41?. The influence of some specific patterns on lipase thermostability was also discussed.     

Two exposed amino acid residues confer thermostability on a cold shock protein

Thermophilic organisms produce proteins of exceptional stability. To understand protein thermostability at the molecular level we studied a pair of cold shock proteins, one of mesophilic and one of thermophilic origin, by systematic mutagenesis. Although the two proteins differ in sequence at 12 positions, two surface-exposed residues are responsible for the increase in stability of the thermophilic protein (by 15.8 kJ mol-1 at 70 degrees C). 11.5 kJ mol-1 originate from a predominantly electrostatic contribution of Arg 3 and 5.2 kJ mol-1 from hydrophobic interactions of Leu 66 at the carboxy terminus. The mesophilic protein could be converted to a highly thermostable form by changing the Glu residues at positions 3 and 66 to Arg and Leu, respectively. The variation of surface residues may thus provide a simple and powerful approach for increasing the thermostability of a protein.     

易错PCR提高华根霉脂肪酶的热稳定性

为了提高华根霉Rhizopus chinensis CCTCC M201021脂肪酶的热稳定性,运用定向进化-易错PCR的方法,经两轮易错PCR引入突变,利用fast-blue RR顶层琼脂法对突变文库进行筛选,第一轮易错PCR后筛选到2株突变菌株,第二轮筛选到4株突变株。第二轮最佳突变株Ep2-4,其中3个氨基酸发生了突变：A129S、P168L和V329A。该突变酶ep2-4在60 ℃下半衰期相对原始酶r27RCL提高5.4倍,T50值提高7.8 ℃。酶学性质研究表明,突变酶ep2-4在热稳定性提高的基础上,仍保有良好的催化活性。蛋白质三维结构模拟显示,突变A129S可以和Gln133形成氢键,增加了酶表面的亲水性和极性;P168L可以与邻近的Leu164形成疏水键,导致突变酶的热稳定性提高。     

The primary structure of Bacillus cereus neutral proteinase and comparison with thermolysin and Bacillus subtilis neutral proteinase

The complete amino-acid sequence of a neutral proteinase, produced by Bacillus cereus, was determined by protein sequencing. The neutral proteinase consists of 317 amino-acid residues. The primary structure is 70% homologous to thermolysin, a thermostable neutral proteinase and 45% homologous to Bacillus subtilis neutral proteinase. The zinc-binding site and the hydrophobic pocket of the active site are highly similar in all three proteinases. B. cereus neutral proteinase which is 20 degrees C less thermostable (60 degrees C) than thermolysin (80 degrees C) shows only minor differences in calcium binding sites and salt bridges compared to thermolysin (known from its X-ray diffraction analysis), whereas B. subtilis neutral proteinase (50 degrees C) differs considerably. Therefore it was assumed that the difference in thermostability between B. cereus neutral proteinase and thermolysin is not caused by different metal binding properties, or differences in the active site, but by changes within the rest of the molecule. Calculation of secondary structure potentials according to Chou & Fasman, hydrophobicity and bulkiness of the different structural elements and preferred cold----hot amino-acid residue exchanges indicated, that the thermostability of thermolysin compared to B. cereus neutral proteinase is caused by small effects contributed by numerous amino-acid exchanges distributed over the whole molecule, resulting in increased hydrophobicity of beta-pleated sheet and higher bulkiness of alpha-helical regions.     

The first crystal structure of hyperthermostable NAD-dependent glutamate dehydrogenase from Pyrobaculum islandicum

The extremely thermostable NAD-dependent glutamate dehydrogenase (NAD-GluDH) from Pyrobaculum islandicum, a member of the Crenarchaeota, was crystallized, and its 3D structure has been determined by X-ray diffraction methods. The homohexameric structure of Pb. islandicum glutamate dehydrogenase (Pis-GluDH) was solved and refined at a resolution of 2.9A with a crystallographic R-factor of 19.9% (Rfree 26.0%). The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site. The secondary structural elements and catalytically important residues of the enzyme were highly conserved among the NAD(P)-dependent GluDHs from other sources. A structural comparison of Pis-GluDH with other NAD(P)-dependent GluDHs suggests that a significant difference in the alpha8-loop-alpha9 region of this enzyme is associated with its coenzyme specificity. From the analysis of the 3D structure, hydrophobic interactions between intersubunits were found to be important features for the enzyme oligomerization. It has been reported that Pis-GluDH is highly thermostable, like the GluDH of the hyperthermophilic archaeum Pyrococcus furiosus, and the increase in the intersubunit ion pair networks is responsible for the extreme thermostability of the Pc. furiosus enzyme. However, the number of intersubunit ion pairs in the Pis-GluDH molecules is much smaller than those of the Pc. furiosus GluDH. The number of hydrophobic interactions at the intersubunit interfaces were increased and responsible for the extremely high thermostability. This indicates that the major molecular strategy for high thermostability of the GluDHs may be different for each hyperthermophile.     

Effects of changing the interaction between subdomains on the thermostability of Bacillus neutral proteases.

Variants of the thermolabile neutral protease (Npr) of B. subtilis (Npr-sub) and the thermostable neutral protease of B. stearothermophilus (Npr-ste) were produced by means of site-directed mutagenesis and the effects of the mutations on thermostability were determined. Mutations were designed to alter the interaction between the middle and C-terminal subdomain of these enzymes. In all Nprs a cluster of hydrophobic contacts centered around residue 315 contributes to this interaction. In thermostable Nprs (like Npr-ste) a 10 residue beta-hairpin, covering the domain interface, makes an additional contribution. The hydrophobic residue at position 315 was replaced by smaller amino acids. In addition, the beta-hairpin was deleted from Npr-ste and inserted into Npr-sub. The changes in thermostability observed after these mutations confirmed the importance of the hydrophobic cluster and of the beta-hairpin for the structural integrity of Nprs. Combined mutants showed that the effects of individual mutations affecting the interaction between the subdomains were not additive. The effects on thermostability decreased as the strength of the subdomain interaction increased. The results show that once the subdomain interface is sufficiently stabilized, additional stabilizing mutations at the same interface do not further increase thermostability. The results are interpreted on the basis of a model for the thermal inactivation of neutral proteases, in which it is assumed that inactivation results from the occurrence of local unfolding processes that render these enzymes susceptible to autolysis.     

Use of amber suppressors to investigate the thermostability of Bacillus licheniformis alpha-amylase. Amino acid replacements at 6 histidine residues reveal a critical position at His-133

A set of 12 Escherichia coli suppressor tRNAs, inserting different amino acids in response to an amber codon, has been used to create rapidly numerous protein variants of a thermostable amylase; by site-directed mutagenesis, amber mutations were first introduced into Bacillus licheniformis alpha-amylase gene at position His35, His133, His247, His293, His406, or His450; genes carrying one or two amber mutations were then expressed in the different suppressor strains, generating over 100 amylase variants with predicted amino acid changes that could be tested for thermostability. Within the detection limits of the assays, amino acid replacements at five histidine positions had no significant effect. In contrast, suppressed variants substituted at residue His133 clearly exhibited modified thermostability and could be either less stable or more stable than the wild-type amylase, depending on the amino acid inserted at this position; comparison of the variants indicates that the hydrophobicity of the substituting residue is an important but not a determinant factor of stabilization. The effect of the most stabilizing and destabilizing amino acid substitutions, His133 to Tyr and to Pro, respectively, were confirmed by introducing the corresponding missense mutations into the gene sequence. The advantages and limits of informational suppression in protein stability studies are discussed as well as structural features involved in the thermostability of B. licheniformis alpha-amylase.     

Spontaneous tandem sequence duplications reverse the thermal stability of carboxyl-terminal modified 3-isopropylmalate dehydrogenase.

A mutant strain of Thermus thermophilus which contains deletions in the 3'-terminal region of its leuB gene showed a temperature-sensitive growth phenotype in the absence of leucine. Three phenotypically thermostable mutants were isolated from the temperature-sensitive strain by spontaneous evolution. Each pseudorevertant carried a tandem sequence duplication in the 3' region of its leuB gene. The mutated 3-isopropylmalate dehydrogenases encoded by the leuB genes from the pseudorevertants were more thermostable than the enzyme from the temperature-sensitive strain. Structural analyses suggested that the decreased thermostability of the enzyme from the temperature-sensitive strain was caused by reducing hydrophobic and electrostatic interactions in the carboxyl-terminal region and that the recovered stability of the enzymes from the pseudorevertants was due to the restoration of the hydrophobic interaction. Our results indicate that tandem sequence duplications are the general genetic way to alter protein characteristics in evolution.     

A rigid network of long-range contacts increases thermostability in a mutant endoglucanase

Thermodynamic stability of a protein at elevated temperatures is a key factor for thermostable enzymes to catalyze their specific reactions. Yet our understanding of biological determinants of thermostability is far from complete. Many different atomistic factors have been suggested as possible means for such proteins to preserve their activity at high temperatures. Among these factors are specific local interatomic interactions or enrichment of specific amino acid types. The case of glycosyl hydrolase family endoglucanase of Trichoderma reesei defies current hypotheses for thermostability because a single mutation far from the active site (A35?V) converts this mesostable protein into a thermostable protein without significant change in the protein structure. This substantial change in enzymatic activity cannot be explained on the basis of local intramolecular interactions alone. Here we present a more global view of the induced thermostability and show that the A35?V mutation affects the underlying structural rigidity of the whole protein via a number of long-range, non-local interactions. Our analysis of this structure reveals a precisely tuned, rigid network of atomic interactions. This cooperative, allosteric effect promotes the transformation of this mesostable protein into a thermostable one.