Effect of Confinement on Protein Conformation in Presence of Osmolytes Urea and Trimethylamine-N-Oxide

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The mechanism by which TMAO provides counteraction against the action of urea and stabilizes protein has been investigated. We focus on the effects of urea and TMAO on the folding/unfolding equilibrium of protein and put an effort to uncover the molecular basis of denaturation and counteraction mechanisms. In Chapter 1, we present a detailed literature review of the mechanism of action of the osmolytes, their effects on the protein. The reason of choosing REMD (Replica Exchange Molecular Dynamics) simulation technique over the classical MD technique is also explained. In Chapters, we have performed a comparative study utilizing Kast and Osmotic models of TMAO to examine the extent of protein protecting ability of the two models. We have tried to explore the underlying mechanism by which urea and TMAO exert their effects on the protein -hairpin. Urea causes denaturation of the protein through breaking of the terminal intra-protein hydrogen bonds. Kast model of TMAO, in conventional 2:1 ratio of urea:TMAO provides counteraction against denaturing effect of urea. Osmotic model provides maximum counteraction when used in 1:2 ratio, but fails to provide sufficient conformational stability to the protein in conventional 2:1 ratio of urea and TMAO. These findings lead us to carry out further studies with Kast model of TMAO. In Chapter 3, we have emphasized on the counteraction behavior of TMAO against action of urea without using any confinement. Using REMD simulation technique we have studied urea-induced denaturation and TMAO induced counteraction of the protein Trp cage. We have particularly emphasized on three factors that persuade stability on the protein Trp cage: (i) salt bridge between residues Asp9 and Arg16, (ii) the buried hydrophobic core surrounding the indole plane of residue Trp6 and (iii) orientational preference of different aromatic planes constituting the protein with respect to the indole plane of residue Trp6. Large number of protein-urea hydrogen bonds indicate direct interaction of urea with the protein that result in unfolding of the protein.
Supervisor: Sandip Paul