Beyond the active site

Abstract

It is important to understand the relationship between protein structure and biochemical function to provide a basis for the effective design of activators or inhibitors of proteins in normal and disease states. There has been a rapid increase in the number of known protein structures and in the techniques for probing their activity but relatively few studies explore the connection between catalytic function and conformational dynamics of proteins. Dynamics are critical to a protein's ability to carry out its function and can include conformational changes, specific domain motions, or fluctuations of individual residues as part of the mechanism. These residues participate in molecular recognition and, in the cases of enzymes, may contribute significantly to catalysis. The dynamics of these residues both near and remote from the active site of an enzyme may participate in catalysis. Presented in this dissertation are four chapters focusing on using small-angle x-ray solution scattering (SAXS) and computational tools to understand the relationship of protein dynamics and function in two proteins, Escherichia coli Ornithine transcarbamoylase (OTC) and human parkin. Both of these proteins were predicted by Partial Order Optimum Likelihood (POOL) to have distal residues that are important for catalysis. The last chapter of this dissertation focuses on using these same tools for drug discovery, specifically on Cu/Zn Superoxide dismutase 1 (SOD1), a protein that is implicated in the onset of Amyotrophic Lateral Sclerosis (ALS). Chapter 2 describes how SAXS was used to understand how distal residues in OTC play a role in dynamical structural changes, thus affecting catalysis from a distance. Three-dimensional shape envelopes of the SAXS data for wild-type OTC and variants provided insight into how distal residues, shown to significantly affect catalysis, undergo a dramatic structural rearrangement. Therefore, in addition to electrostatic effects, these distal residues may play a role in modulating dynamics and thus contributing to the catalytic mechanism of OTC. Chapter 3 describes SAXS experiments on wild-type and mutant constructs of parkin. Three-dimensional shape envelopes from the SAXS data of these different constructs provided a basis for constructing a molecular model of the active conformation of parkin. The attachment of a SUMO-tag to the N-terminus of parkin resulted in an active conformation and provided insight into the dramatic re-positioning of domains. This was the first model of the active conformation of parkin proposed on the basis of structural data collected directly from the full-length form. Chapter 4 describes the use of POOL to identify distal residues remote from the active site of parkin that contribute to catalysis. Interestingly, a subset of these predicted residues are positions in which mutations are implicated in the onset of Parkinson's disease (PD). Enzyme variants were created through single-site directed mutagenesis to probe the contribution of these residues to catalysis on the basis of our model for active parkin described in Chapter 3. Lastly, the Agar group at Northeastern University has shown that cyclic disulfide compounds are promising reagents for stabilizing the dimer form of SOD1. These compounds work by forming a disulfide bond between the Cys111 residues on each subunit of SOD1, located in the dimer interface. Chapter 5 describes how computational tools were used to predict which cyclic disulfide compounds are most likely to stabilize a number of ALS-associated variants of SOD1. These predictions were tested through a set of SAXS and CD experiments that were done to determine the impact of cyclic disulfide compounds that have been shown to form a crosslink on the overall structure of SOD1. These studies provide insight into the stability of SOD1 mutant dimers and identified compounds with the greatest potency for maintaining a compact folded state.