Abstract
Chapter 3 : The stability of BTK SH3 domain has been characterized by equilibrium circular dichroism (CD), and fluorescence. Hydrogen-deuterium exchange experiments and backbone dynamics at four temperatures are also performed to monitor the stability and heat capacity at the level of individual residues. The folding kinetics of BTK SH3 domain is investigated with stopped-flow CD, fluorescence and quenched-flow pulse l ling. Our results indicate that BTK SH3 folds via a two-state process and the formation of secondary and tertiary structure are concurrent. The free energy of unfolding, DGUH2O, estimated from guanidine denaturation is 3.87 ± 0.07 kcal/mol, which is in good agreement with those estimated from thermal denaturation, and kinetic experiments. Hydrogen-deuterium (HD) exchange studies of BTK SH3 indicate that the opening process leading to exchange is highly cooperative and b2, b3, and b4 are the most stable regions for exchange. The free energies of exchange (DGHD) of slowly exchanging protons are higher than the free energy of unfolding (DGUH2O). This may indicates that the denatured state might be more compact than the extended conformation, and it is also confirmed from the fact that the DCp value from thermal denaturation is higher than that from empirical calculation. The contributions to the heat capacity (Cp) from fast backbone dynamics have been determined from the temperature dependence of order parameters. The higher mean Cp values of N-terminal b1 and C-terminal b5 at folded state may contribute to the high denaturation temperature of BTK SH3 domain. Quenched-flow pulse l ling experiments indicate that the slow exchange core is also the folding core. b2, b3 together with b4 forms the folding core, and the following protected regions are the diverging turn and the C-terminal b5. N-terminal b1 and first half of RT loop are protected most slowly. From these studies, we know that the deletion on SH3 domain, which loses b4 and b5, have large effects kinetically and thermodynamically.