Methodologies for the analysis of membrane systems using lipid nanodiscs

Membrane proteins are biologically significant targets of study due to their crucial roles in biochemical reactions, such as ion transport and cell signaling. Their study, however, is hampered by hydrophobic regions in their structures which cause aggregation without the presence of a membrane. For this reason, solubilization systems have been developed, but there are limitations to most. Nanodiscs were developed as an alternative platform that provides a native-like lipid bilayer for solubilizing membrane proteins with unparalleled control over lipid composition, exceptional monodispersity, and exceptional modularity. This dissertation details the coupling of Nanodiscs to multiple analytical platforms for the characterization of membrane systems, including Cytochrome P450s, blood coagulation factor proteins, and other membrane protein targets. The topology of three different Cytochrome P450 systems was characterized using linear dichroism spectroscopy. Methods were developed for the coupling of Nanodiscs to two different types of photonic biosensors, in atmospheric pressure and high pressure environments. The work further details the development of a microfluidic platform for the optimization of membrane protein incorporation into Nanodiscs as well as the development of a bimodal imaging construct utilizing Nanodiscs and a Gd(III) chelating molecule used as a contrast agent for the labeling of cells