Folding studies of outer surface protein A fragments from Borrellia burgdorferi: A model beta-sheet, and, Structural investigation of the catalytic doman of human type II inosine 5'-monophosphate dehydrogenas

The primary amino acid sequence of a protein dictates its overall three-dimensional structure, which arises through discrete folding processes. Outer Surface Protein A (OspA) represents a model system to investigate beta-sheet protein secondary structure formation. Through isolation of a unique single layer beta-sheet (SLB) segment from OspA that lacks tertiary contacts, the propensity for beta-sheet formation was examined in the absence of auxiliary interactions. A series of peptide segments from the SLB of OspA were investigated for conformation, thermodynamic stability, and mobility. These studies provided new insight into the importance of buried hydrophobic surface area and its influence on cooperative folding. Removal of the C-terminal domain of OspA allowed the unique SLB domain to be isolated and indicated this segment was competent to yield a fully folded protein. Analysis using Nuclear Magnetic Resonance Spectroscopy (NMR) generated structural and dynamic information for this N-terminal fragment of OspA and suggested that cooperative strand interactions and edge strand exposure significantly impacted folding. The study of OspA aids understanding of intra-molecular interactions that drive folding, specifically in the beta-sheet conformation.;The inter-molecular interactions that drive specific binding were investigated through Inosine 5'-Monophosphate Dehydrogenase (IMPDH). IMPDH is the rate-limiting enzyme in the de novo synthesis of guanine nucleotides. As such, its inhibition leads to the depletion of guanine nucleotides, which disrupts DNA replication. Rapidly proliferating cells such as leukocytes and leukemic cells are particularly affected, thereby making IMPDH an attractive target for directed inhibition. Although the structure of IMPDH is known, better crystals diffracting to higher resolution should greatly improve existing knowledge of the enzyme active site. Here, a method is described for producing the catalytic domain of IMPDH, which was purified and crystallized for structural studies. Solubilization conditions were obtained by combining fusion-protein expression with sparse matrix screening, yielding a new high-throughput screening method relying on protease cleavage. The effects of chaotropic and molecular crowding agents on the structure of mobile regions of the protein significantly influenced the structure and activity of the enzyme. A new crystal structure refined to 2.45 A resolution reveals key active site amino acids interactions with substrate, co-factors and inhibitors and should lead to further therapeutic developments in IMPDH inhibitors that differentiate clinically relevant IMPDH isoforms. Both intra- and inter molecular interactions contribute to the final structure and function of proteins, the study of which enables better understanding of diseases, and through IMPDH has direct drug design applications.