NMR studies of protein folding of the villin headpiece subdomain

The chicken villin headpiece (HP67) is derived from the F-actin-bundling protein villin. The C-terminal subdomain of HP67 can adopt the native fold in isolation and the isolated C-terminal construct is denoted HP36. My work focuses on folding studies of HP67 and HP36.; Using 1H dynamic NMR line-shape analysis, I demonstrate that HP36 folds on the timescale of microseconds. Folding rates were estimated using resolved protein resonances from three different residues at both 500 MHz and 700 MHz.{09}No significant changes on the folding rates were observed upon mutation of Phe76 despite theoretical studies suggesting that it slows folding.; A set of peptide fragments derived from HP36 were characterized to determine any significant tendency to form locally stabilized structure in the unfolded state. A 21-residue peptide fragment, denoted HP21, shows considerable structure as judged by NMR and CD.{09}Strongly upfield shifted Calpha protons, the magnitude of the 3JNH,alpha coupling constants and the pattern of backbone to backbone and backbone to sidechain NOEs indicate that there is significant secondary structure and hydrophobic clustering in the unfolded state of HP36.; The effect of modulating unfolded state structure on the folding kinetics of the villin headpiece subdomain was investigated. The results demonstrate that the folding time for the villin headpiece subdomain is not critically dependent on the residual structure in the unfolded state that is present in HP36 but diminished in the mutant in which Phe47 and Phe51 are changed to leucine.; The multi-state folding process of HP67 was investigated. NMR, CD and H/D amide exchange measurements were used to follow the pH, thermal and urea induced unfolding of HP67 and a mutant in which His41 has been mutated to Tyr. The result shows that HP67 undergoes pH-dependent segmental unfolding.{09}At the pH where both domains of HP67 are folded, the unfolding is still multi-state with the N-terminal subdomain being less stable than the C-terminal subdomain. The folded N-terminal subdomain significantly stabilizes the C-terminal subdomain. Mutation of His41 to Tyr eliminates the segmental pH dependent unfolding of HP67, but thermal unfolding is still not two-state. The mutation stabilizes both the N and C-terminal subdomains.