Abstract
Molecular basis of protein stability at different temperatures is a fundamental problem in protein science that is substantially far from being accurately and quantitatively solved as it requires an explicit knowledge of the temperature dependence of folding free energy of amino acid residues. In the present study, we attempted to gain insights into the thermodynamic stability of SazCA and its implications on protein folding/unfolding. We report molecular dynamics simulations of water solvated SazCA in a temperature range of 293-393 K to study the relationship between the thermostability and flexibility. Our structural analysis shows that the protein maintains the highest structural stability at 353 K and the protein conformations are highly flexible at temperatures above 353 K. Larger exposure of hydrophobic surface residues to the solvent medium for conformations beyond 353 K were identified from H-bond analysis. Higher number of secondary structure contents exhibited by SazCA at 353 K corroborated the conformations at 353 K to exhibit the highest thermal stability. The analysis of thermodynamics of protein stability revealed that the conformations that denature at higher melting temperatures tend to have greater maximum thermal stability. Our analysis shows that 353 K conformations have the highest melting temperature, which was found to be close to the experimental optimum temperature. The enhanced protein stability at 353 K due the least value of heat capacity at unfolding suggested an increase in folding. Comparative Gibbs free energy analysis and funnel shaped energy landscape confirmed a transition in folding/unfolding pathway of SazCA at 353 K.
Highlights
Carbonic anhydrases (CAs) constitute a family of Zn-containing metalloenzymes which catalyze the reversible hydration of CO2 to bicarbonates and protons [1,2,3,4], as shown in Eq (1).CO2 þ H2O Ð HCOÀ3 þ Hþ ð1ÞThe enzymes of this family are known to exhibit one of the fastest reaction rates known so far in nature [1]
Studies on unfolding pathways of bovine CA and CAIX in association with conformational stability have been reported [23]. Such details on thermostable SazCA are missing and we provide them by quantifying the secondary structure descriptors, H-bonding descriptors, root mean square deviation (RMSD), root mean square fluctuations (RMSD), radius of gyration (RG), percentage of secondary structure contents, solvent accessible surface area (SASA) and pair correlation function (RDF) in this study
A similar observation of significant loss of B-B H-bonds and a larger exposure of hydrophobic surface residues to the solvent beyond 353 K has been reported to be responsible for unfolding of human isoforms of CA [23]
Summary
Carbonic anhydrases (CAs) constitute a family of Zn-containing metalloenzymes which catalyze the reversible hydration of CO2 to bicarbonates and protons [1,2,3,4], as shown in Eq (1).CO2 þ H2O Ð HCOÀ3 þ Hþ ð1ÞThe enzymes of this family are known to exhibit one of the fastest reaction rates known so far in nature [1]. These are enconded by six evolutionarily distinct gene families viz., α, β, γ, δ, z, and η [5, 6]. CAs have been reported as potential biocatalysts for industrial applications in carbon capture, storage and sequestration (CCUS) [13]. These enzymes are of interest as biocatalytic components in biomedical devices such as biosensors and artificial lungs [14]. We have focussed our attention on another α-CA of same CA family, namely SazCA, because of its potential in high temperature biotechnological CCUS applications. We describe the rationale behind the selection of SazCA in the text to follow
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