| Membrane proteins are involved in numerous biological processes, including transport, signal transduction, and a variety of metabolic pathways. Despite their abundance, however, membrane proteins largely remain resistant to biophysical characterization due to complexities in sample preparation and limited knowledge regarding structural elucidation. The overlying objective of the work reported in this dissertation is focused upon developing proteomics based approaches for the structural investigation of integral membrane proteins involved in the vitamin K cycle.;Vitamin K is an essential micronutrient that functions as a coenzyme in the carboxylation of vitamin K-dependent (VKD) proteins by the integral membrane protein Gamma-Glutamyl Carboxylase (GGCX). Concomitant with VKD modification, vitamin K is regenerated by a mechanism involving the enzyme Vitamin K Epoxide Reductase (VKOR) and the cycle continues. Structural analysis of GGCX and VKOR is of particular interest in understanding their functional involvement in blood coagulation, calcification, and cell growth control. Although the mechanisms for carboxylation and epoxidation have been investigated for over thirty years, biological recognition involving structural conformations and protein associations are not yet completely understood.;As an alternative approach to classical biochemical experimentation, methods were developed to investigate GGCX protein dynamics by ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry. Following a brief overview of the vitamin K cycle (Chapter 1), Chapters 2-4 aim to develop analytical approaches for identifying the catalytic active site in GGCX using covalent cross-linking mass spectrometry. A comprehensive bottom-up proteomics methodology (Chapter 2) was applied to identify site-specific covalent attachment of synthetically modified VKD cross-linker substrates in Chapters 3 and 4. In Chapter 5, a new class of model membrane, Nanodiscs, are introduced providing a controlled, native phospholipid structure in which membrane proteins can be isolated in a water-soluble environment. Incorporation of GGCX embedded Nanodiscs are further investigated by hydrogen exchange mass spectrometry (HX MS) in Chapter 6. This novel system demonstrates the first reported application for investigation of membrane protein dynamics in a near-native environment by HX MS. The work outlined in this dissertation not only offers significant advancements in the structural investigation of GGCX, but provides unique platforms in which to investigate other complex membrane protein systems. |
