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Mechanical unfolding and folding studies on proteins with high sequence identity but different conformations Li, Jinliang

Abstract

Although a few mutations can radically shift the equilibrium between denatured state and native state of a protein, it is surprising that one mutation can switch one fold into another completely different fold. Two Streptococcus binding domains GA and GB could be mutated so that ultimately two completely different folds had only one different amino acid in their sequences. This experiment established a mutational pathway to switch a protein’s fold and function. In order to further understand the mechanism underlying this pathway, single molecule force spectroscopy was carried out using optical tweezers to investigate certain proteins along the mutational pathway to determine their mechanical stability and unfolding/folding kinetics. In this dissertation, GB’s homologous protein NuG2 was studied and demonstrated that the force spectroscopy was a robust and informative tool to determine the unfolding/folding kinetics and the free energy profile of protein unfolding. Additionally, the kinetics and free energy profiles of GA and other mutants including GA30, GA77, GA95 and GB30 were characterized. These results provide a clear tendency of free energy change along the mutational pathway.

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Attribution-NonCommercial-NoDerivs 2.5 Canada

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