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Understanding the molecular basis of thermostability and activity of B. subtilis lipase and its mutantsAuthor: Bipin Singh Date: 2017-03-08 Report no: IIIT/TH/2017/11 Advisor:Abhijit Mitra AbstractImproving the thermostability of industrial enzymes is an important protein engineering challenge. Molecular level understanding of mutational effects on stability and activity of enzymes is complex particularly when several point mutations are incorporated during the directed evolution experiments, due to non-additivity involving either cooperative (positive) or antagonistic (negative) effects. Thermostability is experimentally characterized by many observables (T50, Tm, T1/2, ΔG etc.) and they often show correlation with each other. However, this is not always true because of the different stabilizing mechanisms operative in different cases. In this thesis, I have tried to understand the molecular mechanism of thermostability and activity of B. subtilis lipase mutants. I have studied the molecular dynamics of wild type B. subtilis lipase (WT) and its six progressively thermostable mutants (2M, 3M, 4M, 6M, 9M, and 12M), at different temperatures. The less thermostable mutants (LTMs), 2M to 6M, show WT-like dynamics at all simulation temperatures. However, the two more thermostable mutants 9M and 12M (MTMs) show the required flexibility at appropriate temperature ranges and maintain conformational stability even at high temperature. They show a deep and rugged free-energy landscape, confining them within a near-native conformational space by conserving non-covalent interactions, and thus protecting them from possible aggregation. In contrast, the LTMs having marginally higher thermostabilities than WT show greater probabilities of accessing non-native conformations, which, due to aggregation, have reduced possibilities of reverting to their respective native states under refolding conditions. Our analysis indicates the possibility of non-additive effects of point mutations on the conformational stability of LTMs. I suggested the lack of consistency in the effect of point mutations incorporated during the initial generations of directed evolution experiments, towards conformational stabilization of B. subtilis lipase mutants of later generations. I show that the cumulative point mutations incorporated in mutants 2M (with two point mutations) to 6M (with six point mutations) possibly do not retain their original stabilizing nature in the most thermostable 12M mutant (with 12 point mutations). I have carried out MD simulations using structures incorporating reversal of different sets of point mutations to assess their effect on the conformational stability and activity of 12M. My analysis has revealed that reversal of certain point mutations in 12M had little effect on its conformational stability, suggesting that these mutations were probably inconsequential towards the thermostability of the 12M mutant. Interestingly, these mutations involved evolutionarily conserved residues. On the other hand, some of the other point mutations incorporated in non-conserved regions, appeared to contribute significantly towards the conformational stability and/or activity of 12M. Based on the analysis of dynamics of in-silico mutants generated using the consensus sequence, I identified experimentally verifiable residue positions to further increase the conformational stability and activity of the 12M mutant. In this context, I also studied, the protein contact networks (PCNs) and its application in understanding the small conformational variations due to point mutations using both, the crystal structures as well as the MD trajectories of the different mutants. Analysis of the variation in several network centrality measures showed some important correlations with stabilities of different mutants. I have identified key residues, in 9M and 12M, which can be related to their enhanced stability and activity compared to WT and other less thermostable mutants. I have observed that the reversal of few key point mutations in 12M have significant effect on the topological features, suggesting the importance of these point mutations in the stability/activity of 12M. The study of contact networks based on non-covalent interactions effectively bring out the factors responsible for enhanced stability/activity due to point mutations in more thermostable 9M and 12M mutants. Our study highlights the importance of considering not only the geometrical criteria but also the chemistry of interacting amino acids in PCN construction. Analysis of the molecular basis of chain length selectivity in B. subtilis lipase and the role of catalytically important point mutations in the substrate binding of the 12M mutant suggest that the combined reversal of M134E and M137P point mutations cause significant decrease in binding affinity for the small chain length ester substrates compared to long chain length ester substrates, suggesting the importance of M134E and M137P point mutations in substrate binding and hence activity. The 12M mutant maintains a nearly constant volume for binding site with all the substrates and stabilizes a ready to attack conformation with long chain substrates, explaining the molecular basis of its high activity compared to WT and other less thermostable mutants. I propose that in order to further increase the activity of B. subtilis lipase towards longer chain substrates; we need to incorporate residues which provide favorable van der Waals interaction between the long acyl chain and the catalytically important residues such as S77, I12 and M78, through increase in the volume of the binding cleft. In summary, detailed computational investigation discussed in this thesis provides significant insights about the mechanism of point mutations in thermostability, molecular basis for non-additivity and interpretation and explanations of the earlier experimental observations related to thermostability and activity. This thesis will be useful for researchers working in the area of protein science and engineering to efficiently engineer proteins for better thermostability and invoke new and interesting avenues for further research. Full thesis: pdf Centre for Computational Natural Sciences and Bioinformatics |
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