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.     

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.     

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.     

An electrostatic basis for the stability of thermophilic proteins

Two factors provide key contributions to the stability of thermophilic proteins relative to their mesophilic homologues: electrostatic interactions of charged residues in the folded state and the dielectric response of the folded protein. The dielectric response for proteins in a "thermophilic series" globally modulates the thermal stability of its members, with the calculated dielectric constant for the protein increasing from mesophiles to hyperthermophiles. This variability results from differences in the distribution of charged residues on the surface of the protein, in agreement with structural and genetic observations. Furthermore, the contribution of electrostatic interactions to the stability of the folded state is more favorable for thermophilic proteins than for their mesophilic homologues. This leads to the conclusion that electrostatic interactions play an important role in determining the stability of proteins at high temperatures. The interplay between electrostatic interactions and dielectric response also provides further rationalization for the enhanced stability of thermophilic proteins with respect to cold-denaturation. Taken together, the distribution of charged residues and their fluctuations have been shown to be factors in modulating protein stability over the entire range of biologically relevant temperatures.     

Thermodynamic differences among homologous thermophilic and mesophilic proteins.

Here, we analyze the thermodynamic parameters and their correlations in families containing homologous thermophilic and mesophilic proteins which show reversible two-state folding <--> unfolding transitions between the native and the denatured states. For the proteins in these families, the melting temperatures correlate with the maximal protein stability change (between the native and the denatured states) as well as with the enthalpic and entropic changes at the melting temperature. In contrast, the heat capacity change is uncorrelated with the melting temperature. These and additional results illustrate that higher melting temperatures are largely obtained via an upshift and broadening of the protein stability curves. Both thermophilic and mesophilic proteins are maximally stable around room temperature. However, the maximal stabilities of thermophilic proteins are considerably greater than those of their mesophilic homologues. At the living temperatures of their respective source organisms, homologous thermophilic and mesophilic proteins have similar stabilities. The protein stability at the living temperature of the source organism does not correlate with the living temperature of the protein. We tie thermodynamic observations to microscopics via the hydrophobic effect and a two-state model of the water structure. We conclude that, to achieve higher stability and greater resistance to high and low temperatures, specific interactions, particularly electrostatic, should be engineered into the protein. The effect of these specific interactions is largely reflected in an increased enthalpy change at the melting temperature.     

Effective factors in thermostability of thermophilic proteins

Thermostability of proteins in general and especially thermophilic proteins has been subject of a wide variety of studies based on theoretical and experimental investigation. Thermostability seems to be a property obtained through many minor structural modifications rather than certain amino acids substitution. In comparison with its mesophile homologue in a thermostable protein, usually a number of amino acids are exchanged. A wide variety of theoretical studies are based on comparative investigation of thermophilic proteins characteristics with their mesophilic counterparts in order to reveal their sequences, structural differences and consequently, to relate these observed differences to the thermostability properties. In this work we have compared a dataset of thermophilic proteins with their mesophilic homologues and furthermore, a mesophilic proteins dataset was also compared with its mesophilic homologue. This strategy enabled us first, to eliminate noise or background differences from signals and moreover, the important factors which were related to the thermostability were recognized too. Our results reveal that thermophilic and mesophilic proteins have both similar polar and nonpolar contribution to the surface area and compactness. On the other hand, salt bridges and main chain hydrogen bonds show an increase in the majority of thermophilic proteins in comparison to their mesophilic homologues. In addition, in thermophilic proteins hydrophobic residues are significantly more frequent, while polar residues are less. These findings indicate that thermostable proteins through evolution adopt several different strategies to withstand high temperature environments.     

A search for structural factors responsible for the stability of proteins from thermophilic organisms

A database was designed to include 392 pairs of homologous proteins from thermophilic and mesophilic organisms. Proteins from thermophilic organisms proved to contain more atom-atom contacts per residue as compared with their mesophilic homologs. Solvent-accessible exterior amino acid residues contribute to the increase in the number of contacts. The amino acid composition was analyzed for internal (solvent-inaccessible) and exterior amino acid residues of thermophilic and mesophilic proteins. The exterior residues of thermophils have higher contents of Lys, Arg, and Glu and lower contents of Ala, Asp, Asn, Gln, Ser, and Thr as compared with mesophilic proteins. Interior protein regions did not differ in amino acid composition.     

Using motif-based methods in multiple genome analyses: a case study comparing orthologous mesophilic and thermophilic proteins

Protein motifs represent highly conserved regions within protein families and are generally accepted to describe critical regions required for protein stability and/or function. In this comprehensive analysis, we present a robust, unique approach to identify and compare corresponding mesophilic and thermophilic sequence motifs between all orthologous proteins within 44 microbial genomes. Motif similarity is determined through global sequence alignment of mesophilic and thermophilic motif pairs, which are identified by a greedy algorithm. Our results reveal only modest correlation between motif and overall sequence similarity, highlighting the rationale of motif-based approaches in comprehensive multigenome comparisons. Conserved mutations reflect previously suggested physiochemical principles for conferring thermostability. Additionally, comparisons between corresponding mesophilic and thermophilic motif pairs provide key biochemical insights related to thermostability and can be used to test the evolutionary robustness of individual structural comparisons. We demonstrate the ability of our unique approach to provide key insights in two examples: the TATA-box binding protein and glutamate dehydrogenase families. In the latter example, conserved mutations hint at novel origins leading to structural stability differences within the hexamer structures. Additionally, we present amino acid composition data and average protein length comparisons for all 44 microbial genomes.     

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.     

Protein rigidity and thermophilic adaptation

We probe the hypothesis of corresponding states, according to which homologues from mesophilic and thermophilic organisms are in corresponding states of similar rigidity and flexibility at their respective optimal temperatures. For this, the local distribution of flexible and rigid regions in 19 pairs of homologous proteins from meso- and thermophilic organisms is analyzed and related to activity characteristics of the enzymes by constraint network analysis (CNA). Two pairs of enzymes are considered in more detail: 3-isopropylmalate dehydrogenase and thermolysin-like protease. By comparing microscopic stability features of homologues with the help of stability maps, introduced for the first time, we show that adaptive mutations in enzymes from thermophilic organisms maintain the balance between overall rigidity, important for thermostability, and local flexibility, important for activity, at the appropriate working temperature. Thermophilic adaptation in general leads to an increase of structural rigidity but conserves the distribution of functionally important flexible regions between homologues. This finding provides direct evidence for the hypothesis of corresponding states. CNA thereby implicitly captures and unifies many different mechanisms that contribute to increased thermostability and to activity at high temperatures. This allows to qualitatively relate changes in the flexibility of active site regions, induced either by a temperature change or by the introduction of mutations, to experimentally observed losses of the enzyme function. As for applications, the results demonstrate that exploiting the principle of corresponding states not only allows for successful thermostability optimization but also for guiding experiments in order to improve enzyme activity in protein engineering.     

Search for structural factors responsible for the stability of proteins from thermophilic organisms

Database including 392 homologous pairs of proteins from thermophilic and mesophilic organisms was created. Using this database we have found that proteins from termophilic organisms contain more atom-atom contacts per residue in comparison with mesophilic homologues. Contribution to increase of the number of contacts gives exterior amino acid residues, accessible for the solvent. Amino acid composition of interior, inaccessible for the solvent, and exterior amino acid residues of proteins from thermophilic and mesophilic organisms were analyzed. We have obtained that exterior residues of proteins from thermophilic organisms contain more such amino acid residues as Lys, Arg and Glu and smaller such amino acid residues as Ala, Asp, Asn. Gln, Ser, and Thr in comparison with proteins from mesophilic organisms. Amino acid compositions of interior residues of considered proteins are not different.     

Different packing of external residues can explain differences in the thermostability of proteins from thermophilic and mesophilic organisms

MOTIVATION: Understanding the basis of protein stability in thermophilic organisms raises a general question: what structural properties of proteins are responsible for the higher thermostability of proteins from thermophilic organisms compared to proteins from mesophilic organisms? RESULTS: A unique database of 373 structurally well-aligned protein pairs from thermophilic and mesophilic organisms is constructed. Comparison of proteins from thermophilic and mesophilic organisms has shown that the external, water-accessible residues of the first group are more closely packed than those of the second. Packing of interior parts of proteins (residues inaccessible to water molecules) is the same in both cases. The analysis of amino acid composition of external residues of proteins from thermophilic organisms revealed an increased fraction of such amino acids as Lys, Arg and Glu, and a decreased fraction of Ala, Asp, Asn, Gln, Thr, Ser and His. Our theoretical investigation of folding/unfolding behavior confirms the experimental observations that the interactions that differ in thermophilic and mesophilic proteins form only after the passing of the transition state during folding. Thus, different packing of external residues can explain differences in thermostability of proteins from thermophilic and mesophilic organisms. AVAILABILITY: The database of 373 structurally well-aligned protein pairs is available at http://phys.protres.ru/resources/termo_meso_base.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Exploiting the Link between Protein Rigidity and Thermostability for Data‐Driven Protein Engineering

Understanding and exploiting the relationship between microscopic structure and macroscopic stability is important for developing strategies to improve protein stability at high temperatures. The thermostability of proteins has been repeatedly linked to an enhanced structural rigidity of the folded native state. In the current study, the rigidity of protein structures from mesophilic and thermophilic organisms along a thermal unfolding trajectory is directly probed. In order to perform this, protein structures were modeled as constraint networks, and the rigidity in these networks was quantified using the Floppy Inclusion and Rigid Substructure Topography (FIRST) method. During the thermal unfolding, a phase transition was observed that defines the rigidity percolation threshold and corresponds to the folded‐unfolded transition in protein folding. Using concepts from percolation theory and network science, a higher phase transition temperature was observed for ca. two‐thirds of the proteins from thermophilic organisms compared to their mesophilic counterparts, when applied to a data set of 20 pairs of homologues. From both the analysis of the microstructure of the constraint networks and monitoring the macroscopic behavior during the thermal unfolding, direct evidence was found for the “corresponding states” concept, which states that mesophilic and thermophilic enzymes are in corresponding states of similar flexibility at their respective optimal temperature. Finally, the current approach facilitated the identification of structural features from which a destabilization of the structure originates upon thermal unfolding. These predictions show a good agreement with the experimental data. Therefore, the information might be exploited in data‐driven protein engineering by pointing to residues that should be varied to obtain a protein with higher thermostability.     

Importance of main-chain hydrophobic free energy to the stability of thermophilic proteins

Living organisms are found in the most unexpected places, including deep-sea vents at 100 degrees C and several hundred bars pressure, in hot springs. Needless to say, the proteins found in thermophilic species are much more stable than their mesophilic counterparts. There are no obvious reasons to say that one would be more stable than others. Even examination of the amino acids and comparison of structural features of thermophiles with mesophilies cannot bring satisfactory explanation for the thermal stability of such proteins. In order to bring out the hidden information behind the thermal stabilization of such proteins in terms of energy factors and their combinations, analysis were made on good resolution structures of thermophilic and their mesophilic homologous from 23 different families. From the structural coordinates, free energy contributions due to hydrophobic, electrostatic, hydrogen bonding, disulfide bonding and van der Waals interactions are computed. In this analysis, a vast majority of thermophilic proteins adopt slightly lower free energy contribution in each energy terms than its mesophilic counterparts. The major observation noted from this study is the lower hydrophobic free energy contribution due to carbon atoms and main-chain nitrogen atoms in all the thermophilic proteins. The possible combination of different free energy terms shows majority of the thermophilic proteins have lower free energy strategy than their mesophilic homologous. The derived results show that the hydrophobic free energy due to carbon and nitrogen atoms and such combinations of free energy components play a vital role in the thermostablisation of such proteins.     

Different unfolding pathways for mesophilic and thermophilic homologues of serine hydroxymethyltransferase

To determine how much information can be transferred from folding and unfolding studies of one protein to another member of the same family or between the mesophilic and thermophilic homologues of a protein, we have characterized the equilibrium unfolding process of the dimeric enzyme serine hydroxymethyltransferase (SHMT) from two sources, Bacillus subtilis (bsSHMT) and Bacillus stearothermophilus (bstSHMT). Although the sequences of the two enzymes are highly identical ( approximately 77%) and homologous (89%), bstSHMT shows a significantly higher stability against both thermal and urea denaturation than bsSHMT. The GdmCl-induced unfolding of bsSHMT was found to be a two-step process with dissociation of the native dimer, resulting in stabilization of a monomeric species, followed by the unfolding of the monomeric species. A similar unfolding pathway has been reported for Escherichia coli aspartate aminotransferase, a member of the type I fold family of PLP binding enzymes such as SHMT, the sequence of which is only slightly identical ( approximately 14%) with that of SHMT. In contrast, for bstSHMT, a highly cooperative unfolding without stabilization of any monomeric intermediate was observed. These studies suggest that mesophilic proteins of the same structural family even sharing a low level of sequence identity may follow a common unfolding mechanism, whereas the mesophilic and thermophilic homologues of the same protein despite having a high degree of sequence identity may follow significantly different unfolding mechanisms.     

An integrated approach for thermal stabilization of a mesophilic adenylate kinase

Thermally stable proteins are desirable for research and industrial purposes, but redesigning proteins for higher thermal stability can be challenging. A number of different techniques have been used to improve the thermal stability of proteins, but the extents of stability enhancement were sometimes unpredictable and not significant. Here, we systematically tested the effects of multiple stabilization techniques including a bioinformatic method and structure‐guided mutagenesis on a single protein, thereby providing an integrated approach to protein thermal stabilization. Using a mesophilic adenylate kinase (AK) as a model, we identified stabilizing mutations based on various stabilization techniques, and generated a series of AK variants by introducing mutations both individually and collectively. The redesigned proteins displayed a range of increased thermal stabilities, the most stable of which was comparable to a naturally evolved thermophilic homologue with more than a 25° increase in its thermal denaturation midpoint. We also solved crystal structures of three representative variants including the most stable variant, to confirm the structural basis for their increased stabilities. These results provide a unique opportunity for systematically analyzing the effectiveness and additivity of various stabilization mechanisms, and they represent a useful approach for improving protein stability by integrating the reduction of local structural entropy and the optimization of global noncovalent interactions such as hydrophobic contact and ion pairs. Proteins 2014; 82:1947–1959. © 2014 Wiley Periodicals, Inc.     

A comparative molecular dynamics study of thermophilic and mesophilic ribonuclease HI enzymes

We studied a pair of homologous thermophilic and mesophilic ribonuclease HI enzymes by molecular dynamics simulations. Each protein was subjected to three 5 ns simulations in explicit water at both 310 K and 340 K. The thermophilic enzyme showed larger overall positional fluctuations at both temperatures, while only the mesophilic enzyme at the higher temperature showed significant instability. When the temperature is changed, the relative flexibility of different local segments on the two proteins changed differently. Principal component analysis showed that the simulations of the two proteins explored largely overlapping regions in the conformational space. However, at 340 K, the collective structure variations of the thermophilic protein are different from those of the mesophilic protein. Our results, although not in accordance with the view that hyperthermostability of proteins may originate from their conformational rigidity, are consistent with several recent experimental and simulation studies which showed that thermophilic proteins may be conformationally more flexible than their mesophilic counterparts. The decorrelation between conformational rigidity and hyperthermostability may be attributed to the temperature dependence and long range nature of electrostatic interactions that play more important roles in the structural stability of thermophilic proteins.     

Identifying and engineering ion pairs in adenylate kinases. Insights from molecular dynamics simulations of thermophilic and mesophilic homologues

Molecular dynamics simulations were performed to study thermal stabilization of proteins via electrostatic interactions of ion pairs. Dynamic motions of four ion pairs previously proposed to be important in thermal stability of adenylate kinase from the thermophile Bacillus stearothermophilus were monitored during the simulation. One of the four ion pairs identified in the crystal structure, Lys180-Asp114, was not maintained in close contact suggesting that the ion pair does not contribute to thermal stability. Among the other three ion pairs, the ion pair Arg116-Glu198 was proposed to be the most important for stability. To predict behaviors of the ion pairs when engineered into a mesophilic homologue to increase stability, in silico mutants of adenylate kinase from the mesophile Bacillus subtilis were generated, and their molecular dynamics simulations were carried out. The ion pairs in the mutant simulations displayed similar behaviors to those in the simulation of the thermophilic protein. To validate the results of the simulations experimentally, the same mutants were produced in vitro and their thermal stabilities were measured using differential scanning calorimetry. In agreement with the simulations, the Lys180-Asp114 did not result in any increase in stability by itself or additive effect with other ion pairs, whereas a mutant with the Arg116-Glu198 exhibited the highest stability among the mutants having one of the four ion pairs. These results provide specific knowledge about stability in adenylate kinases and more generally suggest that molecular dynamics simulations can provide valuable information for identifying and engineering ion pairs.