| Summary: | Presented in this dissertation are studies on the gas-phase structural features of peptides and peptide fragment ions using mass spectrometry (MS), hydrogen/deuterium (H/D) exchange, infrared multiphoton dissociation (IRMPD) spectroscopy, and computational modeling. Additional studies are presented on the mechanism of hydrogen/deuterium exchange using a model amino acid system. The application of chemical cross-linking to investigate the interaction between two proteins, LexA and RecA, is also presented. Gas-phase structural features can be probed using a number of techniques, and several of the studies presented in this dissertation involve the use of gas-phase H/D exchange. Although the basic mechanism for exchange has been determined, the factors that affect the rate and extent of exchange are not well understood. A computational modeling study of the exchange behavior of asparagine and its methyl ester demonstrated that exchange will occur preferentially at sites of more similar basicity. The distinctive exchange behavior of a model histidine-containing pentapeptide, HAAAA, prompted further studies into the structural features that result in five fast exchanging hydrogens and one slower exchange. Peptide analogues were used to identify the sites of exchange, and IRMPD spectroscopy combined with computational modeling indicated that exchange may occur because interaction with water at those sites results in lower energy structures compared to the other sites. Structural studies were also performed to determine whether the b₂⁺ ion from HAAAA is an oxazolone or diketopiperazine. Although the IRMPD spectrum matched that of a diketopiperazine, H/D exchange and fragmentation studies revealed the presence of both a diketopiperazine and oxazolone structure. Protein-protein interactions perform a vital role in regulating cellular processes. Despite extensive mutational analysis, the binding interaction between LexA and RecA, two proteins involved in the SOS response, is unclear. Chemical cross-linking experiments were undertaken to help target future mutational studies, and these studies identified two possible interactions. The first potential binding interaction is located in the cleft of RecA, and the second interaction may be caused by a LexA dimer binding across the RecA helical groove. The presence of two different binding interactions suggests that LexA may have redundant binding modes for RecA interaction.
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