| Protein-protein interactions regulate essential and diverse biological processes. Identifying and characterizing the interacting surfaces in a complex is essential to comprehending protein-protein complexation. As a first step to identify interacting surfaces, the solvent accessibility of a protein surface was measured by monitoring hydrogen-deuterium exchange (HX) rates of amide protons using mass spectrometry (MS). By proteolytically cleaving the protein prior to analysis, mass changes were localized to specific regions of the protein. Chapter 2 describes a novel technique for monitoring HX rates using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) MS. As is detailed in Chapter 3, those amides located in unstructured regions exchange rapidly and serve as useful probes of solvent accessibility at a protein-protein interface. The interface can be detected by a slowing of the HX rate of these solvent-accessible amides due to decreased solvent accessibility in the complex. Furthermore, the kinetics of solvent accessibility can be determined by measuring the mass change at various solvent-incubation times. In Chapter 4, HX kinetic studies are presented for the thrombin-thrombomodulin interaction at two different pH's. Our hypothesis was that the HX rate of amides that are extremely solvent inaccessible in the complex would not be as pH sensitive as those at the periphery of the interface. Of those amides experiencing reduced solvent accessibility, we found several that were pH insensitive and that are believed to be at the solvent-excluded core of the interface, and several pH-dependent amides that are thought to be at the periphery. The experimental work in this thesis is complemented by a theoretical approach that predicts the binding geometry of two proteins. A docking algorithm that uses convolution methods to score complexes based upon electrostatic and van der Waals energies is described in Chapter 5. As described in Chapter 6, to aid the algorithm with specificity, the HX data was used as an additional potential function in the program in order to help discern true from false positives. |
