This thesis includes work from three major projects. In the first chapter I describe work on the structural heterogeneity of the folding intermediate of RNase H. In this project we were able to populate the kinetic intermediate of RNase H at equilibrium with a mutation that strategically disrupted the native state. By populating this intermediate at equilibrium, we were able to characterize it by NMR and show that it is a highly dynamic conformation. The second chapter presents work using hydrophobic core repacking to manipulate protein function. We used a constrained directed evolution approach to generate novel function in the transcriptional activator MarA. We created libraries of core mutations and selected for core mutants that could stimulate transcription with a novel promoter sequence. Our results demonstrated that reorganization of the core alone can be sufficient to drive the evolution of novel function.;Finally, in the appendix, I describe my work in trying to isolate and characterize a class of mutations in ligand binding proteins which are vitamin remedial. Remedial mutations are those which disrupt protein function, but can be reversed with elevated levels of cofactor. Vitamin remediation is particularly interesting for its therapeutic benefits in the case of mutations linked to heritable disease. We hypothesized that vitamin remedial mutations might be simply derived from shifts in protein stability. To characterize the vitamin remedial effects of mutations in folate-binding proteins, we coupled in vivo evidence for folate-responsive growth to biophysically measured changes in stability and binding.
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