Conferring Thermostability to Mesophilic Proteins through Optimized Electrostatic Surfaces (original) (raw)
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Journal of Chemical Information and Modeling, 2009
In spite of considerable improvement of our understanding of factors responsible for protein thermostability, rational designing of thermostable variants of mesophilic proteins is not yet fully established. The present paper describes an effective computational strategy that we have developed to identify the most suitable mutations converting a chosen mesophilic protein into a thermophilic one starting from its 3D structure. The approach is based on the concept that stabilization of several surface residues should enhance the global stability of the protein. The method relies on the estimation of electrostatic and van der Waals interactions in computing the interaction among the side chains of individual residues and the rest of the protein. The polar or charged residues whose side chains interact weakly with the rest of the original protein are identified first. Then, for each such identified residue (A), another residue (B) in its spatial vicinity is identified. The side chain of the residue (B) is then replaced by a suitable conformer of a residue that is electrostatically complementary to the residue (A) to enhance local interactions and hence the stability of the protein. The steric effect is taken care of through van der Waals interactions. We reject the mutations that improve interactions only locally along the sequence as it is unlikely to enhance the global stability of the 3D architecture. We use the difference in self-energies (∆E self ) as a measure of the stability difference between the original and its mutant variant. This paper presents two test cases with demonstration of the enhanced stability of such mutated proteins and validates the strategy by considering five experimentally known thermophilic-mesophilic protein pairs.
Protein Engineering Design & Selection, 2003
It has been known for some time that thermophilic proteins generally have increased numbers of non-covalent interactions (salt bridges, hydrogen bonds, etc.) compared with their mesophilic orthologs. Recently, anecdotal structural comparisons suggest that non-speci®c acid±base ion pairs on the protein surface can be an evolutionary ef®cient mechanism to increase thermostability. In this comprehensive structural analysis, we con®rm this to be the case. Comparison of 127 orthologous mesophilic± thermophilic protein groups indicates a clear preference for stabilizing acid±base pairs on the surface of thermophilic proteins. Compared with positions in the core, stabilizing surface mutations are less likely to disrupt the tertiary structure, and thus more likely to be evolutionarily selected. Therefore, we believe that our results, in addition to being theoretically interesting, will facilitate identi®cation of charge-altering mutations likely to increase the stability of a particular protein structure.
Extremophiles, 2009
Thermophiles, mesophiles, and psychrophiles have different amino acid frequencies in their proteins, probably because of the way the species adapt to very different temperatures in their environment. In this paper, we analyse how contacts between sidechains vary between homologous proteins from species that are adapted to different temperatures, but displaying relatively high sequence similarity. We investigate whether specific contacts between amino acids sidechains is a key factor in thermostabilisation in proteins. The dataset was divided into two subsets with optimal growth temperatures from 0-40 and 35-102°C. Comparison of homologues was made between low-temperature species and high-temperature species within each subset. We found that unspecific interactions like hydrophobic interactions in the core and solvent interactions and entropic effects at the surface, appear to be more important factors than specific contact types like salt bridges and aromatic clusters.
PLOS One, 2011
Networks and clusters of intramolecular interactions, as well as their ''communication'' across the three-dimensional architecture have a prominent role in determining protein stability and function. Special attention has been dedicated to their role in thermal adaptation. In the present contribution, seven previously experimentally characterized mutants of a cold-adapted a-amylase, featuring mesophilic-like behavior, have been investigated by multiple molecular dynamics simulations, essential dynamics and analyses of correlated motions and electrostatic interactions. Our data elucidate the molecular mechanisms underlying the ability of single and multiple mutations to globally modulate dynamic properties of the cold-adapted a-amylase, including both local and complex unpredictable distal effects. Our investigation also shows, in agreement with the experimental data, that the conversion of the cold-adapted enzyme in a warm-adapted variant cannot be completely achieved by the introduction of few mutations, also providing the rationale behind these effects. Moreover, pivotal residues, which are likely to mediate the effects induced by the mutations, have been identified from our analyses, as well as a group of suitable candidates for protein engineering. In fact, a subset of residues here identified (as an isoleucine, or networks of mesophilic-like salt bridges in the proximity of the catalytic site) should be considered, in experimental studies, to get a more efficient modification of the features of the cold-adapted enzyme.
Hydrophobic environment is a key factor for the stability of thermophilic proteins
Proteins: Structure, Function, and Bioinformatics, 2013
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; 81:715-721. V V C 2012 Wiley Periodicals, Inc.
Many proteins are rapidly deactivated when exposed to high or even ambient temperatures. This cannot only impede the study of a particular protein, but also is one of the major reasons why enzyme catalysis is still widely unable to compete with established chemical processes. Furthermore, differences in protein stability are a challenge in synthetic biology, when individual modules prove to be incompatible. The targeted stabilization of proteins can overcome these hurdles, and protein engineering techniques are more and more reliably supported by computational chemistry tools. Accordingly,
Journal of Molecular Graphics & Modelling, 2007
Adaptation to both high and low temperatures requires proteins with special properties. While organisms living at or close to the boiling point of water need to have proteins with increased stability, other properties are required at temperatures close to the freezing point of water. Indeed, it has been shown that enzymes adapted to cold environments are less resistant to heat with a concomitant increased activity as compared to their warm-active counter-parts. Several recent studies have pointed in the direction that electrostatic interactions play a central role in temperature adaptation, and in this study we investigate the role such interactions have in adaptation of elastase from Atlantic salmon and pig. Molecular dynamics (MD) simulations have been used to generate structural ensembles at 283 and 310 K of the psychrophilic and mesophilic elastase, and a total of eight 12 ns simulations have been carried out. Even though the two homologues have a highly similar three-dimensional structure, the location and number of charged amino acids are very different. Based on the simulated structures we find that very few salt-bridges are stable throughout the simulations, and provide little stabilization/destabilization of the proteins as judged by continuum electrostatic calculations. However, the mesophilic elastase is characterized by a greater number of salt-bridges as well as a putative salt-bridge network close to the catalytic site, indicating a higher rigidity of the components involved in the catalytic cycle. In addition, subtle differences are also found in the electrostatic potentials in the vicinity of the catalytic residues, which may explain the increased catalytic efficiency of the cold-adapted elastase.
Rational Modification of Protein Stability by the Mutation of Charged Surface Residues
Biochemistry, 2000
Continuum methods were used to calculate the electrostatic contributions of charged and polar side chains to the overall stability of a small 41-residue helical protein, the peripheral subunit-binding domain. The results of these calculations suggest several residues that are destabilizing, relative to hydrophobic isosteres. One position was chosen to test the results of these calculations. Arg8 is located on the surface of the protein in a region of positive electrostatic potential. The calculations suggest that Arg8 makes a significant, unfavorable electrostatic contribution to the overall stability. The experiments described in this paper represent the first direct experimental test of the theoretical methods, taking advantage of solid-phase peptide synthesis to incorporate approximately isosteric amino acid substitutions. Arg8 was replaced with norleucine (Nle), an amino acid that is hydrophobic and approximately isosteric, or with R-amino adipic acid (Aad), which is also approximately isosteric but oppositely charged. In this manner, it is possible to isolate electrostatic interactions from the effects of hydrophobic and van der Waals interactions. Both Arg8Nle and Arg8Aad are more thermostable than the wild-type sequence, testifying to the validity of the calculations. These replacements led to stability increases at 52.6°C, the T m of the wild-type, of 0.86 and 1.08 kcal mol -1 , respectively. The stability of Arg8Nle is particularly interesting as a rare case in which replacement of a surface charge with a hydrophobic residue leads to an increase in the stability of the protein.
Journal of Chemical Theory and Computation, 2019
The folding and stability of proteins is a fundamental problem in several research fields. In the present paper, we have used different computational approaches to study the effects caused by changes in pH and for charged mutations in Cold Shock Proteins from Bacillus subtilis (Bs-CspB). First, we have investigated the contribution of each ionizable residue for these proteins on their thermal stability using the TKSA-MC, a Web-server for rational mutation via optimizing the protein charge interactions. Based on these results, we have proposed a new mutation in an already optimized Bs-CspB variant. We have evaluated the effects of this new mutation in the folding energy landscape using Structure-Based models in Monte Carlo simulation at constant pH-SBM-CpHMC. Our results using this approach have indicated that the charge rearrangements already in the unfolded state are critical to the thermal stability of Bs-CspB. Furthermore, the conjunction of these simplified methods was able not only to predict stabilizing mutations in different pHs but also to provide essential information about their effects in each stage of protein folding. 2
Large-scale modulation of thermodynamic protein folding barriers linked to electrostatics
Proceedings of the National Academy of Sciences, 2008
Protein folding barriers, which range from zero to the tens of RT that result in classical two-state kinetics, are primarily determined by protein size and structural topology [Plaxco KW, Simons KT, Baker D (1998) J Mol Biol 277:985-994]. Here, we investigate the thermodynamic folding barriers of two relatively large proteins of the same size and topology: bovine ␣-lactalbumin (BLA) and hen-egg-white lysozyme (HEWL). From the analysis of differential scanning calorimetry experiments with the variable-barrier model [Muñ oz V, Sanchez-Ruiz JM (2004) Proc Natl Acad Sci USA 101: 17646 -17651] we obtain a high barrier for HEWL and a marginal folding barrier for BLA. These results demonstrate a remarkable tuning range of at least 30 kJ/mol (i.e., five to six orders of magnitude in population) within a unique protein scaffold. Experimental and theoretical analyses on these proteins indicate that the surprisingly small thermodynamic folding barrier of BLA arises from the stabilization of partially unfolded conformations by electrostatic interactions. Interestingly, there is clear reciprocity between the barrier height and the biological function of the two proteins, suggesting that the marginal barrier of BLA is a product of natural selection. Electrostatic surface interactions thus emerge as a mechanism for the modulation of folding barriers in response to special functional requirements within a given structural fold.