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Probing the stability of a model jellyroll [beta]-sandwich protein Everts, Sarah

Abstract

A major challenge in the field of protein folding involves dissecting the thermodynamic and kinetic pathways by which a polypeptide acquires its unique, minimal free-energy structure. While much research has focussed on α-helical containing proteins, the folding pathways of their all-β-sheet counterparts remains less well defined. To this end, thermodynamic investigations of the second N-terminal cellulose binding domain of Cellulomonas fimi endoglucanase C (CBDN2) were carried out. This 153 residue domain is composed of 11 β-strands that assemble into two anti-parallel β-sheets with the topology of a jellyroll β-sandwich. Using circular dichroism (CD) spectroscopy, CBDN2 is shown to undergo fully reversible unfolding induced by both heating and the addition of denaturants. Unfolding of CBDN2 occurs in a largely pH independent manner. This thesis describes the results of "native state hydrogen exchange (HX)" experiments in non-denaturing concentrations of GuanidineDCl (GuDCl). Amide hydrogen to deuteron exchange provides a means of obtaining residue specific stability, since these rates reflect the structural fluctuations and hydrogen bond disruptions required for solvent exposure. Many of these rates increase when measured in the presence of low GuDCl concentration, since the free-energy barrier against structural fluctuations is decreased, although the protein remains globally folded. The core residues of CBDN2 exhibit a high degree of protection, exchanging only as a result of global unfolding fluctuations. The stability measured for these unfolding fluctuations corresponds to the global stability measured by CD denaturation. Amide groups in loop regions and near the termini exhibited much less protection, exchanging via low energy fluctuations. There appeared to be no difference in exchange patterns or residue specific stability between residues in sheet-A and sheet-B, suggesting a two-state folding mechanism and the absence of any partially unfolded intermediates. Residues most protected from HX were highly conserved among the 8 most sequence homologous proteins.

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