| This thesis summarizes the work I performed from over the course of five years (2004 to 2009). It provides the reader with the most up to date bibliographic information regarding fatty acid regulation of plasmin/metalloproteinases (MMP) system. Additionally, the studies presented here characterize fatty acid and phosphocholine binding to kringle domains at the molecular level to set the groundwork for an understanding of the basis of regulation.;The first chapter provides a general background for understanding coagulation and fibrinolysis. It highlights the importance of studying fibrinolysis and describes the role of the plasminogen/plasmin (Pgn/Plm) system in other physiological and disease related processes such as cancer. This chapter also provides a basis for understanding the regulation of Pgn at the molecular level via ligand binding to kringle domains.;In the second chapter, the solution structure of Pgn/K5 is presented. First, the chapter explains how 1H and 15N resonance assignments for each protein amino acid residue were used to obtain distance and angle restraints to calculate the K5 structure, to characterize protein-ligand interactions, and, more specifically, to map the AMCHA and OA binding site on K5. Structural binding site features are analyzed to explain the binding specificity of this domain. Finally, important amino acid residues for ligand binding are inferred from a combination of in silico docking and NMR experiments.;Chapter 3 describes fluorescence titration studies performed on individual Pgn kringles and Pgn fragments. Fatty acid binding kringle domains are identified and their respective affinity quantified. Additionally, phosphocholine binding kringles are identified via tryptophan intrinsic fluorescence experiments. This chapter also describes the characterization of phosphocholine-kringle interaction via NMR experiments. Furthermore, a different approach for binding site mapping is explained and validated via protein magnetic relaxation property changes upon ligand binding.;The final chapter summarizes the results and discusses the implications of fatty acid and phosphocholine binding to kringles. Additionally, it suggests directions for future work. |
