Modeling and Engineering Proteins Thermostability

Enzymes have evolved during millions of years to become efficient catalysts for specific biochemical reactions within a specific range of working conditions in the cellular environment. The activity of an enzyme is directly related to its folded structure and even slight changes in its 3D conformation may cause irreversible, negative effects on its activity. The structure of enzymes are very sensitive to the environmental conditions and changes from their optimal conditions, like higher temperature, salt concentration, and pH, might result in denaturation and subsequently inactivation. In the past decades, natural enzymes extracted from different organisms have found a wide range of applications in the industrial and biotechnological setting. For the majority of the applications their activity at high temperatures is more favorable, however enzymes have evolved, in most of the cases, to optimally work in limited range of temperature of their native cellular environment. Therefore, enhancing enzyme thermostability will not only increase their application range, but could also shed light into new aspects of their evolution and chemical activity. The physical chemical principles underlying enzymatic thermostability are keys in fact to understand the way evolution has shaped proteins to adapt to a broad range of temperatures. Understanding the molecular determinants at the basis of protein thermostability, using both in silico methods and in vitro experiments, is also an important way for engineering more thermo-resistant enzymes to be used in the industrial setting, as for instance DNA ligases, which are important for DNA replication and repair and have been long used in molecular biology and biotechnology. In this thesis I used in silico techniques, like molecular modeling and simulation coupled with bioinformatics analyses, to assess existing methods and predict potential thermo-stabilizing mutations for target proteins. First, I studied a thermophilic protein and after exploring the origins of its thermostability I proposed mutations to further increase its thermostability. Then, I took advantage of what learned from this study to explore further thermostability engineering methods in order to develop faster, accurate, and easy-to-use methods that can be generally used for a broad array of proteins. 1. Understanding and engineering thermostability in the DNA ligase from Thermococcus sp. 1519 (LigTh1519). In this thesis, I first addressed the origin of thermostability in the thermophilic DNA ligase from archaeon Thermococcus sp. 1519, and identified thermo-sensitive regions using molecular modeling and simulations. In addition, I predicted mutations that can enhance thermostability of the enzyme through bioinformatics analyses. I showed that thermo-sensitive regions of this enzyme are stabilized at higher temperatures by optimization of charged groups on the surface, and predicted that thermostability can be further increased by further optimization of the network among these charged groups. Engineering this DNA ligase by introducing selected mutations (i.e., A287K, G304D, S364I and A387K) produced eventually a significant and additive increase in the half-life time of the enzyme when compared to the wild-type. Then, based on what I learned from thermostability analyses and improvement of LigTh1519, my aim was to design a general-purpose protein thermostability engineering protocol that can enable thermostability engineering [...]

Dal Peraro, Matteo
Lausanne, EPFL
Other identifiers:
urn: urn:nbn:ch:bel-epfl-thesis6637-4

 Record created 2015-06-09, last modified 2018-01-28

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