残基相互作用网络特征与木聚糖酶耐热性的关系

残基相互作用网络是体现蛋白质中残基与残基之间协同和制约关系的重要形式。残基相互作用网络的拓扑性质以及社团结构与蛋白质的功能和性质有密切的关系。本文在构建一系列耐热木聚糖酶和常温木聚糖酶的残基相互作用网络后,通过计算网络的度、聚类系数、连接强度、特征路径长度、接近中心性、介数中心性等拓扑参数来确定网络拓扑结构与木聚糖酶耐热性的关系。识别残基相互作用网络的hub点,分析hub点的亲疏水性、带电性以及各种氨基酸在hub点中所占的比例。进一步使用GA-Net算法对网络进行社团划分,并计算社团的规模、直径和密度。网络的高平均度、高连接强度、以及更短的最短路径等表明耐热木聚糖酶残基相互作用网络的结构更加紧密;耐热木聚糖酶网络中的hub节点比常温木聚糖酶网络hub节点具有更多的疏水性残基,hub点中Phe、Ile、Val的占比更高。社团检测后发现,耐热木聚糖酶网络拥有更大的社团规模、较小的社团直径和较大的社团密度。社团规模越大表明耐热木聚糖酶的氨基酸残基更倾向于形成大的社团,而较小的社团直径和较大的社团密度则表明社团内部氨基酸残基的相互作用比常温木聚糖酶更强。     

残基相互作用网络特征与木聚糖酶耐热性的关系

残基相互作用网络是体现蛋白质中残基与残基之间协同和制约关系的重要形式。残基相互作用网络的拓扑性质以及社团结构与蛋白质的功能和性质有密切的关系。本文在构建一系列耐热木聚糖酶和常温木聚糖酶的残基相互作用网络后,通过计算网络的度、聚类系数、连接强度、特征路径长度、接近中心性、介数中心性等拓扑参数来确定网络拓扑结构与木聚糖酶耐热性的关系。识别残基相互作用网络的hub点,分析hub点的亲疏水性、带电性以及各种氨基酸在hub点中所占的比例。进一步使用GA-Net算法对网络进行社团划分,并计算社团的规模、直径和密度。网络的高平均度、高连接强度、以及更短的最短路径等表明耐热木聚糖酶残基相互作用网络的结构更加紧密;耐热木聚糖酶网络中的hub节点比常温木聚糖酶网络hub节点具有更多的疏水性残基,hub点中Phe、Ile、Val的占比更高。社团检测后发现,耐热木聚糖酶网络拥有更大的社团规模、较小的社团直径和较大的社团密度。社团规模越大表明耐热木聚糖酶的氨基酸残基更倾向于形成大的社团,而较小的社团直径和较大的社团密度则表明社团内部氨基酸残基的相互作用比常温木聚糖酶更强。     

通过分析来自Pyrococcus abyssi的腈水解酶中带电基团位置变化探讨蛋白耐热机制

蛋白含带电侧链基团的氨基酸的位置对蛋白质的热稳定性有很大的影响,目前,往往采用蛋白质结构模型"静态"分析这一影响。本文以1株耐热的腈水解酶作为研究对象,构建并异源表达了该酶,对其酶学性质进行初步分析。利用Discovery Studio对该酶进行同源建模,得到空间结构。将此空间结构利用Gromacs软件在不同温度下进行分子动力学(MD)模拟,使用Macrodox软件计算蛋白质所有带电氨基酸残基的包埋率与溶剂可接触性面积(SA),发现在不同温度下,部分氨基酸带电基团的SA值会发生显著变化。说明通过静态分析并不一定能够得到非常准确的结果,而应进行动态分析。     

基于残基相互作用网络比对的木聚糖酶热稳定性的研究

研究木聚糖酶的耐热机理能够提升工业生产效率和经济效益。本文使用提出的SI-MAGNA残基相互作用网络比对算法对来自嗜热子囊菌（Thermoascus aurantiacus）的耐热型木聚糖酶和来自变铅青链霉菌（Streptomyces lividans）的常温型木聚糖酶进行网络比对,以探究影响两者结构稳定性和热稳定性的因素。通过比对结果分析：证实了(βα)8-桶结构的低序列保守性及β折叠对结构稳定性的作用;发现了1E0W的loop1区域中独有一个310-螺旋结构影响了结构稳定性;1TUX中的α1’短螺旋结构和相对较短的β4α4-loop区域有助于提升其结构稳定性和酶的热稳定性;推测1TUX的β4α4-loop区域中独有的氢键转折结构和1E0W的loop6区域中独有的2个β桥结构可能引起空间结构的细微差异,从而影响酶的热稳定性。     

蛋白质相互作用热点残基的预测北大核心CSCD

氨基酸突变扫描实验揭示了在蛋白质相互作用的结合过程中大部分的结合自由能是由极少数热点残基贡献的,通常定义结合自由能变化△△G≥2.0 kcal/mol的蛋白质残基为热点残基。热点残基对蛋白质相互作用具有重要意义。因此,如何有效进行热点残基的预测,仍然是一个研究课题。综合蛋白质氨基酸理化属性的加权疏水性、加权残基接触数、结构属性溶剂可接近面积和残基突出指数等特征,提出利用机器学习支持向量机算法来预测热点残基的方法。所提方法在丙氨酸热力学数据库数据和结合界面数据库选定的数据集上有很好的效果。在一定程度上对以后的研究发展有所帮助。     

嗜热与嗜常温微生物的蛋白质氨基酸组成比较

嗜热微生物的嗜热特性与其蛋白质的高度热稳定性紧密相关。为了探索嗜热蛋白质的热稳定机制,比较嗜热和嗜常温微生物的蛋白质在氨基酸组成上的差别,收集110对分别来自嗜热和嗜常温微生物的同源蛋白质序列,比较两组蛋白质各种氨基酸含量以及疏水性氨基酸组成、疏水性指数和荷电氨基酸组成的差别,结果两者在多种氨基酸含量上存在微小但统计学上显著的差别,嗜热蛋白质比嗜常温蛋白质具有较高的平均疏水性和荷电氨基酸组成。对两组蛋白质的“脂肪族氨基酸指数”进行分析,证明嗜热蛋白质之所以具有较高的脂肪族氨基酸指数是由于其亮氨酸含量较高,与影响该指数的其它几种氨基酸无关;从而认为该指数的意义值得怀疑。通过对大量同源嗜热蛋白质和嗜常温蛋白质氨基酸组成的比较,能够揭示一些有关蛋白质热稳定性的普遍规律。     

非解朊栖热菌HG102耐热β-糖苷酶的结构与功能研究

非解朊栖热菌HG10 2耐热 β-糖苷酶为 (β/α)₈桶状结构 ,是具有水解功能和转糖苷功能的单体酶。该酶可以作为一个很好的模型来研究糖苷酶的反应机制、底物特异性和耐热的分子基础。根据对该酶的晶体结构解析和同家族酶的结构比较 ,推测Glu164和Glu338分别是质子供体和亲核基团两个活性位点 ;在α-螺旋N端第一位的脯氨酸和蛋白质外周的精氨酸是耐热机制的关键位点和关键氨基酸残基。为确定这些氨基酸残基的功能 ,通过基因定点突变的方法分别把Glu164、Glu338、Pro316、Pro356、Pro344和Arg325置换成Gln、Ala、Gly、Ala、Phe和Leu ,同时还对Pro316和Pro356进行了双置换。突变酶经过纯化得到电泳纯 ,用CD光谱进行了野生酶和突变酶的结构比较。通过突变酶的酶功能和酶学性质分析 ,结果表明Glu164和Glu338分别是质子供体和亲核基团 ,亲核基团的突变酶TnglyE338A可以合成混合型糖苷键寡糖类似物 ;在α-螺旋N端第一位的Pro316和Pro356以及在蛋白质外周形成离子键的Arg325均是对耐热性有贡献的关键氨基酸残基。     

枯草杆菌蛋白酶嗜热性改造单突变效果评判方法研究

对蛋白质进行嗜热性改造是蛋白质工程的主要问题之一,残基突变方法被广泛运用于其中。本文以枯草杆菌蛋白酶（SUBTILISIN BPN＇）为研究对象,旨在建立评判嗜热性改造效果的方法,选取了有可靠实验资料的9个突变点,运用分子动力学模拟方法,在四种不同模拟条件下,对其中的6个突变体和1个野生型蛋白进行了多种参量的对比分析,提取4个特征有效参量,建立了蛋白酶嗜热性改造单突变效果评判方法;利用该方法对其它3个突变效果进行评判,评判结果与实验资料完全吻合,证明该方法可用于枯草杆菌蛋白酶嗜热性改造单突变效果的评判。     

动态残基网络社团视角下的木聚糖酶耐热性分析

木聚糖酶在食品、饲料、造纸和纺织等行业有重要的应用,研究木聚糖酶的耐热性有助于挖掘其潜在的应用领域并提高经济价值。本文通过采用Louvain算法,对来自变铅青链霉菌的常温木聚糖酶(xyna_strli)和嗜热子囊菌的耐热木聚糖酶(xyna_theau)的动态残基相互作用网络进行社团检测,并分析社团演化与木聚糖酶耐热性的关系。结果表明:常温木聚糖酶的末端存在稳定相互作用;以GLU37和LEU5为中心的残基结构稳固了酶的结构。耐热木聚糖酶中,α8′、α8″上的相互作用提升了酶的热稳定性。通过分析稳定社团发现,稳定社团包含酶的末端通过稳定相互作用提高耐热性。相较于常温木聚糖酶,耐热木聚糖酶的稳定社团维持了更多短螺旋和柔性区域的稳定,其在活性位点GLU237附近的残基减少了活性位点与底物接触,提升了稳定性。     

基于频繁项集挖掘和连续帧间差分的脂肪酶时-空结构模式与耐热性的关系研究

酶的热稳定性问题一直是蛋白质工程领域关注的重点。本研究通过枯草芽孢杆菌脂肪酶的不同模拟温度下的分子动力学模拟轨迹,分析野生型脂肪酶(WTL)及其突变体(6B)残基间相互作用对热稳定性的影响。首先确定残基间的空间关系,采用空间聚类和FP-growth算法,识别出保持协同运动的β3-β8以及C端部分区域,即确定刚性区;接着运用连续帧间差分法确定波动较大的柔性区,发现其主要位于转角处。以300 K常温状态下识别出的刚性区和柔性区为基础,考察刚性区和柔性区在不同温度下相互作用的动态变化规律,发现WTL和6B的刚性区内的相互作用几乎不随温度发生变化,在高温400 K时,WTL柔性区的稳定氢键数急剧减少为7,6B柔性区的稳定氢键数随温度变化较为稳定。另外,6B的柔性区较WTL少了3₁₀螺旋,这是由于突变后的Ser15与突变后的Ser17形成强大的氢键作用,A15S和F17S突变改善了脂肪酶结构的柔性使其热稳定性增强。     

Electrostatic contributions to the stability of a thermophilic cold shock protein

The thermophilic Bacillus caldolyticus cold shock protein (Bc-Csp) differs from the mesophilic Bacillus subtilis cold shock protein B (Bs-CspB) in 11 of the 66 residues. Stability measurements of Schmid and co-workers have implicated contributions of electrostatic interactions to the thermostability. To further elucidate the physical basis of the difference in stability, previously developed theoretical methods that treat electrostatic effects in both the folded and the unfolded states were used in this paper to study the effects of mutations, ionic strength, and temperature. For 27 mutations that narrow the difference in sequence between Bc-Csp and Bs-CspB, calculated changes in unfolding free energy (Delta G) and experimental results have a correlation coefficient of 0.98. Bc-Csp appears to use destabilization of the unfolded state by unfavorable charge-charge interactions as a mechanism for increasing stability. Accounting for the effects of ionic strength and temperature on the electrostatic free energies in both the folded and the unfolded states, explanations for two important experimental observations are presented. The disparate ionic strength dependences of Delta G for Bc-Csp and Bs-CspB were attributed to the difference in the total charges (-2e and -6e, respectively). A main contribution to the much higher unfolding entropy of Bs-CspB was found to come from the less favorable electrostatic interactions in the folded state. These results should provide insight for understanding the thermostability of other thermophilic proteins.     

Thermal stability and atomic-resolution crystal structure of the Bacillus caldolyticus cold shock protein

The bacterial cold shock proteins are small compact beta-barrel proteins without disulfide bonds, cis-proline residues or tightly bound cofactors. Bc-Csp, the cold shock protein from the thermophile Bacillus caldolyticus shows a twofold increase in the free energy of stabilization relative to its homolog Bs-CspB from the mesophile Bacillus subtilis, although the two proteins differ by only 12 out of 67 amino acid residues. This pair of cold shock proteins thus represents a good system to study the atomic determinants of protein thermostability. Bs-CspB and Bc-Csp both unfold reversibly in cooperative transitions with T(M) values of 49.0 degrees C and 77.3 degrees C, respectively, at pH 7.0. Addition of 0.5 M salt stabilizes Bs-CspB but destabilizes Bc-Csp. To understand these differences at the structural level, the crystal structure of Bc-Csp was determined at 1.17 A resolution and refined to R=12.5% (R(free)=17.9%). The molecular structures of Bc-Csp and Bs-CspB are virtually identical in the central beta-sheet and in the binding region for nucleic acids. Significant differences are found in the distribution of surface charges including a sodium ion binding site present in Bc-Csp, which was not observed in the crystal structure of the Bs-CspB. Electrostatic interactions are overall favorable for Bc-Csp, but unfavorable for Bs-CspB. They provide the major source for the increased thermostability of Bc-Csp. This can be explained based on the atomic-resolution crystal structure of Bc-Csp. It identifies a number of potentially stabilizing ionic interactions including a cation-binding site and reveals significant changes in the electrostatic surface potential.     

Electrostatic stabilization of a thermophilic cold shock protein.

The cold shock protein Bc-Csp from the thermophile Bacillus caldolyticus differs from its mesophilic homolog Bs-CspB from Bacillus subtilis by 15.8 kJ mol(-1) in the Gibbs free energy of denaturation (DeltaG(D)). The two proteins vary in sequence at 12 positions but only two of them, Arg3 and Leu66 of Bc-Csp, which replace Glu3 and Glu66 of Bs-CspB, are responsible for the additional stability of Bc-Csp. These two positions are near the ends of the protein chain, but close to each other in the three-dimensional structure. The Glu3Arg exchange alone changed the stability by more than 11 kJ mol(-1). Here, we elucidated the molecular origins of the stability difference between the two proteins by a mutational analysis. Electrostatic contributions to stability were characterized by measuring the thermodynamic stabilities of many variants as a function of salt concentration. Double and triple mutant analyses indicate that the stabilization by the Glu3Arg exchange originates from three sources. Improved hydrophobic interactions of the aliphatic moiety of Arg3 contribute about 4 kJ mol(-1). Another 4 kJ mol(-1) is gained from the relief of a pairwise electrostatic repulsion between Glu3 and Glu66, as in the mesophilic protein, and 3 kJ mol(-1) originate from a general electrostatic stabilization by the positive charge of Arg3, which is not caused by a pairwise interaction. Mutations of all potential partners for an ion pair within a radius of 10 A around Arg3 had only marginal effects on stability. The Glu3-->Arg3 charge reversal thus optimizes ionic interactions at the protein surface by both local and global effects. However, it cannot convert the coulombic repulsion with another Glu residue into a corresponding attraction. Avoidance of unfavorable coulombic repulsions is probably a much simpler route to thermostability than the creation of stabilizing surface ion pairs, which can form only at the expense of conformational entropy.     

Origins of the high stability of an in vitro-selected cold-shock protein

In previous work, we had identified stabilized forms of the cold-shock protein Bs-CspB from Bacillus subtilis in a combinatorial library by an in vitro selection procedure. In this library, the sequence positions 2, 3, 46, 64, 66, and 67 had been randomized, because Bs-CspB differs from the naturally thermostable homolog Bc-Csp from Bacillus caldolyticus, among others, at these six positions. For the most stable selected variant, the midpoint of thermal unfolding (tM) increased by 28.2 deg. C and the Gibbs free energy of unfolding (deltaG(D)) by 19 kJ/mol. Here, we analyzed by site-directed mutagenesis how the selected residues contribute individually to this strong stabilization. Val3 and Val66, which replace Glu3 and Glu66 of wild-type Bs-CspB, each contribute about 7 kJ/mol to stability, the Thr64Arg substitution contributes 4.5 kJ/mol, and 3.2 kJ/mol originate from the Ala46Leu replacement. Gly67 at the carboxy terminus is unimportant for stability, the Arg selected at position 2 is overall slightly destabilizing but improves the coulombic interactions. The best variant differs from Bc-Csp at all six positions; nevertheless, natural and in vitro selection followed similar principles. In both cases, negatively charged residues at the adjacent positions 3 and 66 are avoided, and a positively charged residue is introduced into this area of the protein surface. Its exact location is unimportant. It can be at position 3, as in the thermophilic Bc-Csp, or at positions 2 or 64, as in the most stable selected variant. These positively charged residues contribute to stability not by engaging in pairwise coulombic interactions with a specific carboxyl group, but by generally improving the charge distribution in this particular region of the protein surface. These coulombic effects contribute significantly to the thermostability of the cold-shock proteins. They are only weakly interdependent and best explained by the presence of a flexible ion network at the protein surface. Our results emphasize that surface positions are very good candidates for optimizing protein stability.     

In-vitro selection of highly stabilized protein variants with optimized surface

Thermostable proteins are of prime importance in protein science, but it has remained difficult to develop general strategies for stabilizing a protein. Site-directed mutagenesis based on comparisons with thermophilic homologs is rarely successful because the sequence differences are too numerous and dominated by neutral mutations. Here we used a method of directed evolution to increase the stability of a mesophilic protein, the cold shock protein Bs-CspB from Bacillus subtilis. It differs from its thermophilic counterpart Bc-Csp from Bacillus caldolyticus at 12 surface-exposed positions. To elucidate the stabilizing potential of exposed amino acid residues, six of these variant positions were randomized by saturation mutagenesis, the corresponding library of sequences was inserted into the gene-3-protein of the filamentous phage fd, and stabilized variants were selected by the Proside technique. Proside links the increased protease resistance of stabilized protein variants with the infectivity of the phage. Many strongly stabilized variants of Bs-CspB were identified in two selections, one in the presence of a denaturant and the other at elevated temperature. Several of them are significantly more stable than the naturally thermostable homolog Bc-Csp, and the best variant reaches Tm-Csp (the homolog from the hyperthermophile Thermotoga maritima) in stability. Remarkably, this variant differs from Tm-Csp at five and from Bc-Csp at all six randomized positions. This indicates that proteins can be strongly stabilized by many different sets of surface mutations, and Proside selects them efficiently from large libraries. The course of the selection could be directed by the conditions. In an ionic denaturant non-polar surface interactions were optimized, whereas at elevated temperature variants with improved electrostatics were selected, pointing to two different strategies for stabilization at protein surfaces.     

Impacts of the charged residues mutation S48E/N62H on the thermostability and unfolding behavior of cold shock protein: insights from molecular dynamics simulation with Gō model

The cold shock protein from the hyperthermophile Thermotoga maritima (Tm-Csp) exhibits significantly higher thermostability than its homologue from the thermophile Bacillus caldolyticus (Bc-Csp). Experimental studies have shown that the electrostatic interactions unique to Tm-Csp are responsible for improving its thermostability. In the present work, the favorable charged residues in Tm-Csp were grafted into Bc-Csp by a double point mutation of S48E/N62H, and the impacts of the mutation on the thermostability and unfolding/folding behavior of Bc-Csp were then investigated by using a modified Gō model, in which the electrostatic interactions between charged residues were considered in the model. Our simulation results show that this Tm-Csp-like charged residue mutation can effectively improve the thermostability of Bc-Csp without changing its two-state folding mechanism. Besides that, we also studied the unfolding kinetics and unfolding/folding pathway of the wild-type Bc-Csp and its mutant. It is found that this charged residue mutation obviously enhanced the stability of the C-terminal region of Bc-Csp, which decreases the unfolding rate and changes the unfolding/folding pathway of the protein. Our studies indicate that the thermostability, unfolding kinetics and unfolding/folding pathway of Bc-Csp can be artificially changed by introducing Tm-Csp-like favorable electrostatic interactions into Bc-Csp.

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Graphical abstract Tertiary structure of wild-type cold shock protein from the thermophile Bacillus caldolyticus

     

Thermodynamics of a diffusional protein folding reaction

The folding reactions of several proteins are well described as diffusional barrier crossing processes, which suggests that they should be analyzed by Kramers' rate theory rather than by transition state theory. For the cold shock protein Bc-Csp from Bacillus caldolyticus, we measured stability and folding kinetics, as well as solvent viscosity as a function of temperature and denaturant concentration. Our analysis indicates that diffusional folding reactions can be treated by transition state theory, provided that the temperature and denaturant dependence of the solvent viscosity is properly accounted for, either at the level of the measured rate constants or of the calculated activation parameters. After viscosity correction the activation barriers for folding become less enthalpic and more entropic. The transition from an enthalpic to an entropic folding barrier with increasing temperature is, however, apparent in the data before and after this correction. It is a consequence of the negative activation heat capacity of refolding, which is independent of solvent viscosity. Bc-Csp and its mesophilic homolog Bs-CspB from Bacillus subtilis differ strongly in stability but show identical enthalpic and entropic barriers to refolding. The increased stability of Bc-Csp originates from additional enthalpic interactions that are established after passage through the activated state. As a consequence, the activation enthalpy of unfolding is increased relative to Bs-CspB.     

Role of the chain termini for the folding transition state of the cold shock protein.

Residues Arg3 and Leu66 are crucially important for the enhanced stability of the cold shock protein Bc-Csp from the thermophile Bacillus caldolyticus relative to its homologue Bs-CspB from the mesophile Bacillus subtilis. Arg3, which replaces Glu3 of Bs-CspB, accounts for two-thirds of the stability difference and for the entire difference in Coulombic interactions between the two proteins. Leu66, which replaces Glu66 of Bs-CspB, contributes additional hydrophobic interactions. To elucidate the role of these two residues near the chain termini for the rapid folding of the cold shock proteins, we performed an extensive mutational analysis of the folding kinetics to characterize interactions between residues 3, 46, and 66 in the transition state of folding. We employed a pressure-jump apparatus which allows folding to be followed over a broad range of temperatures and urea concentrations in the time range of microseconds to minutes. The N-terminal region folds early, and the interactions that originate from residue 3 are present to a large extent in the transition state already. They include a hydrophobic contribution, a general electrostatic stabilization by the positive charge of Arg3 in Bc-Csp, and a pairwise Coulombic repulsion with Glu46 in the Arg3Glu variant. The C-terminus appears to be largely unfolded in the transition state. The interactions of Leu66, including those with the already structured N-terminal region, are established only after passage through the transition state. The N- and C-termini of the cold shock proteins thus contribute differently to the folding kinetics, although they are very close in space in the folded protein.     

Optimized variants of the cold shock protein from in vitro selection: structural basis of their high thermostability

The bacterial cold shock proteins (Csp) are widely used as models for the experimental and computational analysis of protein stability. In a previous study, in vitro evolution was employed to identify strongly stabilizing mutations in Bs-CspB from Bacillus subtilis. The best variant found by this approach contained the mutations M1R, E3K and K65I, which raised the midpoint of thermal unfolding of Bs-CspB from 53.8 degrees C to 83.7 degrees C, and increased the Gibbs free energy of stabilization by 20.9 kJ mol(-1). Another selected variant with the two mutations A46K and S48R was stabilized by 11.1 kJ mol(-1). To elucidate the molecular basis of these stabilizations, we determined the crystal structures of these two Bs-CspB variants. The mutated residues are generally well ordered and provide additional stabilizing interactions, such as charge interactions, additional hydrogen bonds and improved side-chain packing. Several mutations improve the electrostatic interactions, either by the removal of unfavorable charges (E3K) or by compensating their destabilizing interactions (A46K, S48R). The stabilizing mutations are clustered at a contiguous surface area of Bs-CspB, which apparently is critically important for the stability of the beta-barrel structure but not well optimized in the wild-type protein.     

Thermophilicity of wild type and mutant cold shock proteins by molecular dynamics simulation

In the attempt to clarify possible mechanisms underlying thermal stability of proteins, we study through molecular dynamics thermophile Bc-Csp, mesophile Bs-CspB, and selected mutants. These proteins have been extensively characterized experimentally; researchers showed that differential thermostability among the wild type proteins is fundamentally linked to one or two mutated amino acids, and that the nature of the effect is electrostatic. They also inferred an atomistic mechanism related to removal of unfavorable interactions, rather than to the formation of salt bridges. Molecular dynamics allows us to confirm and support both hypotheses. Several other collective parameters have also been monitored in relation to thermophilicity, such as global and local rigidity, permanence and number of hydrogen bonds, or of salt links. None of these clearly correlates with the thermal stability of the presently studied proteins.