Comparative Arrhenius plots of enzyme activity from penicillium

Comparison of the Arrhenius plots of three enzymes, formyltetrahydrofolate synthetase, glutathione reductase (GSSGR) and chorismate mutase (CM) from a thermophilic (Penicillium duponti) and a mesophilic (Penicillium chrysogenum) fungus reveals a fairly consistent pattern. In general, those enzymes extracted from mesophiles had lower activation energies than similar enzymes extracted from thermophiles. One enzyme studied, mesophilic glutathione reductase, exhibited a break in its Arrhenius plot. The allosteric enzyme studied showed slightly different sensitivities in the thermophilic versus the mesophilic extracts.     

Discrimination of mesophilic and thermophilic proteins using machine learning algorithms

Discriminating thermophilic proteins from their mesophilic counterparts is a challenging task and it would help to design stable proteins. In this work, we have systematically analyzed the amino acid compositions of 3075 mesophilic and 1609 thermophilic proteins belonging to 9 and 15 families, respectively. We found that the charged residues Lys, Arg, and Glu as well as the hydrophobic residues, Val and Ile have higher occurrence in thermophiles than mesophiles. Further, we have analyzed the performance of different methods, based on Bayes rules, logistic functions, neural networks, support vector machines, decision trees and so forth for discriminating mesophilic and thermophilic proteins. We found that most of the machine learning techniques discriminate these classes of proteins with similar accuracy. The neural network-based method could discriminate the thermophiles from mesophiles at the five-fold cross-validation accuracy of 89% in a dataset of 4684 proteins. Moreover, this method is tested with 325 mesophiles in Xylella fastidosa and 382 thermophiles in Aquifex aeolicus and it could successfully discriminate them with the accuracy of 91%. These accuracy levels are better than other methods in the literature and we suggest that this method could be effectively used to discriminate mesophilic and thermophilic proteins.     

Role of cation-pi interactions to the stability of thermophilic proteins

Elucidating the factors responsible for exhibiting extreme thermal stability of thermophilic proteins is very important for an understanding of the mechanism of protein stability, as well as to design stable proteins. In this work, we have analyzed the influence of cation-pi interactions to enhance the stability from mesophilic to thermophilic proteins. The favorable residue pairs forming such a system of interactions have been brought out. We found that the Tyr has a greater number of such interactions with Lys in thermophilic proteins. Specifically, the same Lys would experience a greater number of cation-pi interactions with several Tyr residues in thermophiles. On the other hand, the influence of Phe in making cation-pi interactions is higher in mesophiles than in thermophiles. Further, a network of cation-pi interactions are maintained by Lys in thermophiles, whereas Arg plays a major role in mesophilic proteins. Moreover, atoms that have a substantial positive charge in both Lys and Arg make a more significant contribution for cation-pi interactions than do cationic group atoms.     

Role of loop dynamics in thermal stability of mesophilic and thermophilic adenylosuccinate synthetase: a molecular dynamics and normal mode analysis study

Enzymes from thermophiles are poorly active at temperatures at which their mesophilic homologs exhibit high activity and attain corresponding active states at high temperatures. In this study, comparative molecular dynamics (MD) simulations, supplemented by normal mode analysis, have been performed on an enzyme Adenylosuccinate synthetase (AdSS) from E. coli (mesophilic) and P. horikoshii (thermophilic) systems to understand the effects of loop dynamics on thermal stability of AdSS. In mesophilic AdSS, both ligand binding and catalysis are facilitated through the coordinated movement of five loops on the protein. The simulation results suggest that thermophilic P. horikoshii preserves structure and catalytic function at high temperatures by using the movement of only a subset of loops (two out of five) for ligand binding and catalysis unlike its mesophilic counterpart in E. coli. The pre-arrangement of the catalytic residues in P. horikoshii is well-preserved and salt bridges remain stable at high temperature (363K). The simulations suggest a general mechanism (including pre-arrangement of catalytic residues, increased polar residue content, stable salt bridges, increased rigidity, and fewer loop movements) used by thermophilic enzymes to preserve structure and be catalytically active at elevated temperatures.     

Pressure stabilization is not a general property of thermophilic enzymes: the adenylate kinases of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii.

The application of 50-MPa pressure did not increase the thermostabilities of adenylate kinases purified from four related mesophilic and thermophilic marine methanogens. Thus, while it has been reported that some thermophilic enzymes are stabilized by pressure (D. J. Hei and D. S. Clark, Appl. Environ. Microbiol. 60:932-939, 1994), hyperbaric stabilization is not an intrinsic property of all enzymes from deep-sea thermophiles.     

Electrostatic strengths of salt bridges in thermophilic and mesophilic glutamate dehydrogenase monomers

Here we seek to understand the higher frequency of occurrence of salt bridges in proteins from thermophiles as compared to their mesophile homologs. We focus on glutamate dehydrogenase, owing to the availability of high resolution thermophilic (from Pyrococcus furiosus) and mesophilic (from Clostridium symbiosum) protein structures, the large protein size and the large difference in melting temperatures. We investigate the location, statistics and electrostatic strengths of salt bridges and of their networks within corresponding monomers of the thermophilic and mesophilic enzymes. We find that many of the extra salt bridges which are present in the thermophilic glutamate dehydrogenase monomer but absent in the mesophilic enzyme, form around the active site of the protein. Furthermore, salt bridges in the thermostable glutamate dehydrogenase cluster within the hydrophobic folding units of the monomer, rather than between them. Computation of the electrostatic contribution of salt bridge energies by solving the Poisson equation in a continuum solvent medium, shows that the salt bridges in Pyrococcus furiosus glutamate dehydrogenase are highly stabilizing. In contrast, the salt bridges in the mesophilic Clostridium symbiosum glutamate dehydrogenase are only marginally stabilizing. This is largely the outcome of the difference in the protein environment around the salt bridges in the two proteins. The presence of a larger number of charges, and hence, of salt bridges contributes to an electrostatically more favorable protein energy term. Our results indicate that salt bridges and their networks may have an important role in resisting deformation/unfolding of the protein structure at high temperatures, particularly in critical regions such as around the active site.     

Subunit interfaces of oligomeric hyperthermophilic enzymes display enhanced compactness

Enzymes from thermophilic and, particularly, from hyperthermophilic organisms are surprisingly stable. Understanding the molecular origin of protein thermostability and thermoactivity attracted the interest of many scientists both for the perspective comprehension of the principles of protein structure and for the possible biotechnological applications through protein engineering. Comparative studies at sequence and structure levels were aimed at detecting significant differences of structural parameters related to protein stability between thermophilic and hyperthermophilic proteins and their mesophilic homologs. In a recent work, we focused attention on structural adaptation occurring at the subunit interface of oligomeric hyper- and thermostable enzymes. A set of structural and chemico-physical parameters were compared to those observed at the corresponding interfaces of homologous mesophilic proteins. Among the most significant variations, a general increase of interface apolarity and packing density in hyperthermophilic enzymes were found. This work was therefore aimed at elucidating whether the increased packing observed is reached also through the reduction of interface cavity number and volume. The results indicate that number of cavities tends to be relatively constant while cavity volume tends to decrease in the hyperthermophilic interfaces. The cavity apolarity increases in thermophiles but, apparently, not in hyperthermophiles. Moreover, interface hot spot residues of the mesophilic interfaces tend to be conserved in the extremophilic counterparts.     

Hydrophobic environment is a key factor for the stability of thermophilic proteins

The stability of thermophilic proteins has been viewed from different perspectives and there is yet no unified principle to understand this stability. It would be valuable to reveal the most important interactions for designing thermostable proteins for such applications as industrial protein engineering. In this work, we have systematically analyzed the importance of various interactions by computing different parameters such as surrounding hydrophobicity, inter‐residue interactions, ion‐pairs and hydrogen bonds. The importance of each interaction has been determined by its predicted relative contribution in thermophiles versus the same contribution in mesophilic homologues based on a dataset of 373 protein families. We predict that hydrophobic environment is the major factor for the stability of thermophilic proteins and found that 80% of thermophilic proteins analyzed showed higher hydrophobicity than their mesophilic counterparts. Ion pairs, hydrogen bonds, and interaction energy are also important and favored in 68%, 50%, and 62% of thermophilic proteins, respectively. Interestingly, thermophilic proteins with decreased hydrophobic environments display a greater number of hydrogen bonds and/or ion pairs. The systematic elimination of mesophilic proteins based on surrounding hydrophobicity, interaction energy, and ion pairs/hydrogen bonds, led to correctly identifying 95% of the thermophilic proteins in our analyses. Our analysis was also applied to another, more refined set of 102 thermophilic–mesophilic pairs, which again identified hydrophobicity as a dominant property in 71% of the thermophilic proteins. Further, the notion of surrounding hydrophobicity, which characterizes the hydrophobic behavior of residues in a protein environment, has been applied to the three‐dimensional structures of elongation factor‐Tu proteins and we found that the thermophilic proteins are enriched with a hydrophobic environment. The results obtained in this work highlight the importance of hydrophobicity as the dominating characteristic in the stability of thermophilic proteins, and we anticipate this will be useful in our attempts to engineering thermostable proteins. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Pressure Stabilization of Proteins from Extreme Thermophiles

We describe the stabilization by pressure of enzymes, including a hydrogenase from Methanococcus jannaschii, an extremely thermophilic deep-sea methanogen. This is the first published report of proteins from thermophiles being stabilized by pressure. Inactivation studies of partially purified hydrogenases from an extreme thermophile (Methanococcus igneus), a moderate thermophile (Methanococcus thermolithotrophicus), and a mesophile (Methanococcus maripaludis), all from shallow marine sites, show that pressure stabilization is not unique to enzymes isolated from high-pressure environments. These studies suggest that pressure stabilization of an enzyme may be related to its thermophilicity. Further experiments comparing the effects of increased pressure on the stability of α-glucosidases from the hyperthermophile Pyrococcus furiosus and Saccharomyces cerevisiae support this possibility. We have also examined pressure effects on several highly homologous glyceraldehyde-3-phosphate dehydrogenases from mesophilic and thermophilic sources and a rubredoxin from P. furiosus. The results suggest that hydrophobic interactions, which have been implicated in the stabilization of many thermophilic proteins, contribute to the pressure stabilization of enzymes from thermophiles.     

Patterns of temperature adaptation in proteins from the bacteria Deinococcus radiodurans and Thermus thermophilus

Asymmetrical patterns of amino acid substitution in proteins of organisms living at moderate and high temperatures (mesophiles and thermophiles, respectively) are generally taken to indicate selection favoring different amino acids at different temperatures due to their biochemical properties. If that were the case, comparisons of different pairs of mesophilic and thermophilic taxa would exhibit similar patterns of substitutional asymmetry. A previous comparison of mesophilic versus thermophilic Methanococcus with mesophilic versus thermophilic Bacillus revealed several pairs of amino acids for which one amino acid was favored in thermophilic Bacillus and the other was favored in thermophilic Methanococcus. Most of this could be explained by the higher G+C content of the DNA of thermophilic Bacillus, a phenomenon not seen in the Methanococcus comparison. Here, I compared the mesophilic bacterium Deinococcus radiodurans and its thermophilic relative Thermus thermophilus, which are similar in G+C content. Of the 190 pairs of amino acids, 83 exhibited significant substitutional asymmetry, consistent with the pervasive effects of selection. Most of these significantly asymmetrical pairs of amino acids were asymmetrical in the direction predicted from the Methanococcus data, consistent with thermal adaptation resulting from universal biochemical properties of the amino acids. However, 12 pairs of amino acids exhibited asymmetry significantly different from and in the opposite direction of that found in the Methanococcus comparison, and 21 pairs of amino acids exhibited asymmetry that was significantly different from that found in the Bacillus comparison and could not be explained by the greater G+C content in thermophilic Bacillus. This suggests that selection due to universal biochemical properties of the amino acids and differences in G+C content are not the only causes of substitutional asymmetry between mesophiles and thermophiles. Instead, selection on taxon-specific properties of amino acids, such as their metabolic cost, may play a role in causing asymmetrical patterns of substitution.     

Important inter-residue contacts for enhancing the thermal stability of thermophilic proteins

Proteins from thermophilic organisms exhibit high thermal stability, but have structures that are very similar to their mesophilic homologues. In order to gain insight into the basis of thermostability, we have analyzed the medium- and long-range contacts in mesophilic and thermophilic proteins of 16 different families. We found that the thermophiles prefer to have contacts between residues with hydrogen-bond-forming capability. Apart from hydrophobic contacts, more contacts are observed between polar and non-polar residues in thermophiles than mesophiles. Residue-wise analysis showed that Tyr has good contacts with several other residues, and Cys has considerably higher long-range contacts in thermophiles compared with mesophiles. Furthermore, the residues occurring in the range of 31-34 residues apart in the sequence contribute significant long-range contacts to the stability of thermophilic proteins.     

Elucidation of factors responsible for enhanced thermal stability of proteins: a structural genomics based study

Understanding the molecular basis for the enhanced stability of proteins from thermophiles has been hindered by a lack of structural data for homologous pairs of proteins from thermophiles and mesophiles. To overcome this difficulty, complete genome sequences from 9 thermophilic and 21 mesophilic bacterial genomes were aligned with protein sequences with known structures from the protein data bank. Sequences with high homology to proteins with known structures were chosen for further analysis. High quality models of these chosen sequences were obtained using homology modeling. The current study is based on a data set of models of 900 mesophilic and 300 thermophilic protein single chains and also includes 178 templates of known structure. Structural comparisons of models of homologous proteins allowed several factors responsible for enhanced thermostability to be identified. Several statistically significant, specific amino acid substitutions that occur going from mesophiles to thermophiles are identified. Most of these are at solvent-exposed sites. Salt bridges occur significantly more often in thermophiles. The additional salt bridges in thermophiles are almost exclusively in solvent-exposed regions, and 35% are in the same element of secondary structure. Helices in thermophiles are stabilized by intrahelical salt bridges and by an increase in negative charge at the N-terminus. There is an approximate decrease of 1% in the overall loop content and a corresponding increase in helical content in thermophiles. Previously overlooked cation-pi interactions, estimated to be twice as strong as ion-pairs, are significantly enriched in thermophiles. At buried sites, statistically significant hydrophobic amino acid substitutions are typically consistent with decreased side chain conformational entropy.     

Using a strategy based on the concept of convergent evolution to identify residue substitutions responsible for thermal adaptation

Factors that are related to thermostability of proteins have been extensively studied in recent years, especially by comparing thermophiles and mesophiles. However, most of them are global characters. It is still not clear how to identify specific residues or fragments which may be more relevant to protein thermostability. Moreover, some of the differences among the thermophiles and mesophiles may be due to phylogenetic differences instead of thermal adaptation. To resolve these problems, I adopted a strategy to identify residue substitutions evolved convergently in thermophiles or mesophiles. These residues may therefore be responsible for thermal adaptation. Four classes of genomes were utilized in this study, including thermophilic archaea, mesophilic archaea, thermophilic bacteria, and mesophilic bacteria. For most clusters of orthologous groups (COGs) with sequences from all of these four classes of genomes, I can identify specific residues or fragments that may potentially be responsible for thermal adaptation. Functional or structural constraints (represented as sequence conservation) were suggested to have higher impact on thermal adaptation than secondary structure or solvent accessibility does. I further compared thermophilic archaea and mesophilic bacteria, and found that the most diverged fragments may not necessarily correspond to the thermostability-determining ones. The usual approach to compare thermophiles and mesophiles without considering phylogenetic relationships may roughly identify sequence features contributing to thermostability; however, to specifically identify residue substitutions responsible for thermal adaptation, one should take sequence evolution into consideration.     

The specific activities of mesophilic and thermophilic proteinases

1. Most enzymes from extreme thermophiles do not possess higher specific activities than similar enzymes from mesophiles (measured at their respective growth temperatures). 2. However, using protein substrates, the specific activities of thermophilic proteinases are considerably higher than those of most microbial and eukaryotic proteinases. 3. This property could be attributed to purely kinetic influences on the enzyme, to some specific "design" feature of the proteinase, or to the effects of temperature on the substrate. 4. Comparisons of the rates of hydrolysis of large and small substrates by both mesophilic and thermophilic proteinases suggest that temperature-induced changes in substrate susceptibility are a major factor.     

A meta-analysis of the activity,stability, and mutational characteristics of temperature-adapted enzymes

Understanding the characteristics that define temperature-adapted enzymes has been a major goal of extremophile enzymology in recent decades. In the present study, we explore these characteristics by comparing psychrophilic, mesophilic, and thermophilic enzymes. Through a meta-analysis of existing data, we show that psychrophilic enzymes exhibit a significantly larger gap (T_g) between their optimum and melting temperatures compared with mesophilic and thermophilic enzymes. These results suggest that T_g may be a useful indicator as to whether an enzyme is psychrophilic or not and that models of psychrophilic enzyme catalysis need to account for this gap. Additionally, by using predictive protein stability software, HoTMuSiC and PoPMuSiC, we show that the deleterious nature of amino acid substitutions to protein stability increases from psychrophiles to thermophiles. How this ultimately affects the mutational tolerance and evolutionary rate of temperature adapted organisms is currently unknown.     

Protein surface amino acid compositions distinctively differ between thermophilic and mesophilic bacteria

One of the well-known observations of proteins from thermophilic bacteria is the bias of the amino acid composition in which charged residues are present in large numbers, and polar residues are scarce. On the other hand, it has been reported that the molecular surfaces of proteins are adapted to their subcellular locations, in terms of the amino acid composition. Thus, it would be reasonable to expect that the differences in the amino acid compositions between proteins of thermophilic and mesophilic bacteria would be much greater on the protein surface than in the interior. We performed systematic comparisons between proteins from thermophilic bacteria and mesophilic bacteria, in terms of the amino acid composition of the protein surface and the interior, as well as the entire amino acid chains, by using sequence information from the genome projects. The biased amino acid composition of thermophilic proteins was confirmed, and the differences from those of mesophilic proteins were most obvious in the compositions of the protein surface. In contrast to the surface composition, the interior composition was not distinctive between the thermophilic and mesophilic proteins. The frequency of the amino acid pairs that are closely located in the space was also analyzed to show the same trend of the single amino acid compositions. Interestingly, extracellular proteins from mesophilic bacteria showed an inverse trend against thermophilic proteins (i.e. a reduced number of charged residues and rich in polar residues). Nuclear proteins from eukaryotes, which are known to be abundant in positive charges, showed different compositions as a whole from the thermophiles. These results suggest that the bias of the amino acid composition of thermophilic proteins is due to the residues on the protein surfaces, which may be constrained by the extreme environment.     

Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey

BACKGROUND: Proteins from thermophilic organisms usually show high intrinsic thermal stability but have structures that are very similar to their mesophilic homologues. From prevous studies it is difficult to draw general conclusions about the structural features underlying the increased thermal stability of thermophilic proteins. RESULTS: In order to reveal the general evolutionary strategy for changing the heat stability of proteins, a non-redundant data set was compiled comprising all high-quality structures of thermophilic proteins and their mesophilic homologues from the Protein Data Bank. The selection (quality) criteria were met by 64 mesophilic and 29 thermophilic protein subunits, representing 25 protein families. From the atomic coordinates, 13 structural parameters were calculated, compared and evaluated using statistical methods. This study is distinguished from earlier ones by the strict quality control of the structures used and the size of the data set. CONCLUSIONS: Different protein families adapt to higher temperatures by different sets of structural devices. Regarding the structural parameters, the only generally observed rule is an increase in the number of ion pairs with increasing growth temperature. Other parameters show just a trend, whereas the number of hydrogen bonds and the polarity of buried surfaces exhibit no clear-cut tendency to change with growth temperature. Proteins from extreme thermophiles are stabilized in different ways to moderately thermophilic ones. The preferences of these two groups are different with regards to the number of ion pairs, the number of cavities, the polarity of exposed surface and the secondary structural composition.     

Bacterial elongation factor Ts: isolation and reactivity with elongation factor Tu.

An improved method for the purification of bacterial polypeptide elongation factor Ts (EF-Ts) from one mesophile (Escherichia coli) and two thermophiles (Bacillus stearothermophilus and PS3) is described. The improvements are both in the facility of isolation and in increased yields. The purified factors were used for cross-reactivity studies with elongation factor Tu (EF-Tu) obtained from the same bacterial strains. In all combinations studied, the efficiency of EF-Ts in catalyzing the exchange of EF-Tu-bound GDP was proportional to the strength of the protein-protein complex. Whereas the factors from the two thermophiles were interchangeable, the mesophilic EF-Ts formed a very weak complex with thermophilic EF-Tu; however, thermophilic EF-Ts formed very strong complexes with mesophilic EF-Tu. Thus, e.g., EF-Tu from E. coli formed a complex with EF-Ts from B. stearothermophilus which was 10 times more stable than the corresponding homologous complex.     

Aromatic clusters: a determinant of thermal stability of thermophilic proteins

A number of factors have been elucidated as responsible for the thermal stability of thermophilic proteins. However, the contribution of aromatic interactions to thermal stability has not been systematically studied. In the present investigation we used a graph spectral method to identify aromatic clusters in a dataset of 24 protein families for which the crystal structures of both the thermophilic and their mesophilic homologues are known. Our analysis shows a presence of additional aromatic clusters or enlarged aromatic networks in 17 different thermophilic protein families, which are absent in the corresponding mesophilic homologue. The additional aromatic clusters identified in the thermophiles are smaller in size and are largely found on the protein surface. The aromatic clusters are found to be relatively rigid regions of the surface and often the additional aromatic cluster is located close to the active site of the thermophilic enzyme. The residues in the additional aromatic clusters are preferably mutated to Leu, Ser or Ile in the mesophilic homologue. An analysis of the packing geometry of the pairwise aromatic interaction in the additional aromatic clusters shows a preference for a T-shaped orthogonal packing geometry. The present study also provides new insights for protein engineers to design thermostable and thermophilic proteins.     

How Do Thermophilic Proteins and Proteomes Withstand High Temperature?

We attempt to understand the origin of enhanced stability in thermophilic proteins by analyzing thermodynamic data for 116 proteins, the largest data set achieved to date. We compute changes in entropy and enthalpy at the convergence temperature where different driving forces are maximally decoupled, in contrast to the majority of previous studies that were performed at the melting temperature. We find, on average, that the gain in enthalpy upon folding is lower in thermophiles than in mesophiles, whereas the loss in entropy upon folding is higher in mesophiles than in thermophiles. This implies that entropic stabilization may be responsible for the high melting temperature, and hints at residual structure or compactness of the denatured state in thermophiles. We find a similar trend by analyzing a homologous set of proteins classified based only on the optimum growth temperature of the organisms from which they were extracted. We find that the folding free energy at the temperature of maximal stability is significantly more favorable in thermophiles than in mesophiles, whereas the maximal stability temperature itself is similar between these two classes. Furthermore, we extend the thermodynamic analysis to model the entire proteome. The results explain the high optimal growth temperature in thermophilic organisms and are in excellent quantitative agreement with full thermal growth rate data obtained in a dozen thermophilic and mesophilic organisms.