The goal of this proposal is to understand how the amino acid sequence of a protein dictates its fold and stability. In order to accomplish this goal, we need to understand the structure and stability of all the conformations accessible to a given sequence, i.e. its energy landscape. The difficult with this is that proteins are extremely cooperative. In order to bypass this problem, we have dissected a protein's structure and stability into smaller pieces and studied both the partially-folded conformations and folded parts of the protein E. coli ribonuclease H. by comparing the results from kinetic and equilibrium experiments, we have obtained evidence for a surprising conclusion: all of the partially folded conformations (the acid molten globule, the higher energy conformations of the native state and a kinetic folding intermediate) are structured in the same specific regions of the protein. RNase H folds in an apparently hierarchical fashion in which the most stable individual element folds first. These results suggest important implications about the relationship between protein stability, structure and folding. The experiments outlined in this proposal explore the implications of this work to learn what makes one region of the protein the most stable and how the overall energy landscape of a protein affects its global stability, folding and dynamics. Specifically, the aims of this proposal are: 1. Explore the energy landscape of E. coli RNase H using site-specific mutagenesis, testing the relationship between the hierarchy of stability in the native state and the folding pathways(s) of the protein. 2. Determine the structure, stability and folding of autonomous folding fragments comprising the folding core of RNase H. 3. Determine the energy landscape of a thermophilic RNase H (T. thermophilus). The feature of the structure, stability and folding of this protein will be compare to the mesophilic homologue to see if the general rules for thermostability lie in the distribution of energy within the protein.