| A recombinant DNA vector is created to express genetically engineered mutant and wild-type RNase A in chapter one. The purpose of producing mutant forms of RNase A is to make analogues to explore different aspects of the complex folding pathway of this protein. A methodology is developed to express and purify mutant and wild-type RNase A. Physical chemical characterization of all four three-disulfide mutants of RNase A in terms of activity, thermal stability, and spectroscopic properties is performed.;Destabilizing effects resulting from the loss of the Cys40-Cys95 disulfide bond in RNase A are explored further in chapter three. Thermodynamic comparisons reveal a large difference in the global unfolding free energies between wild-type and (C40A, C95A) RNase A (;In chapter two, an analog of the major rate-determining intermediate in the oxidative regeneration pathway of RNase A is characterized in terms of regular backbone structure and thermodynamic stability at 20... |
