Dynamics and Interactions of Membrane Proteins

Membrane proteins are members of the class of proteins that perform their functions while being associated with a lipid bilayer. In the cell, they serve as transporters, receptors, anchors and enzymes. The domain organisation of these proteins suggests importance of lipid membrane and protein-lipid interactions for protein function. The requirement of a membrane mimic and the level of its resemblance to a native one for protein investigation makes the studies of membrane proteins a challenging project. My research work is focusing on the biophysical and biochemical studies of membrane proteins. This dissertation outlines two separate projects, each with their own challenges. Ras proteins are members of a superfamily of small GTPases that act as molecular switches that are involved in signal transduction pathways responsible for cell division and proliferation and, as one might guess, protein malfunction can lead to cancer. Recently, there have been a number of studies that suggest Ras protein dimerization on lipid membranes through protein-protein interactions between G- domains. On the basis of the results obtained from solution NMR and fluorescence polarization anisotropy studies, we concluded that the G-domain of the Ras protein by itself is not prone to dimerization. The result of this work was later confirmed by publications from other groups that performed studies in the presence of the lipid bilayer. NADPH-cytochrome P450 oxidoreductase (POR) is an integral membrane protein involved in an electron transport pathway transferring electrons from NADPH to cytochrome P450. The goal was achieved by application of lipid nanodisc technology, 13C-methyl extrinsic labeling coupled with Methyl-TROSY NMR technique that resulted in signals that showed differential sensitivity towards the redox state of the protein cofactors and conformational transitions of the protein. Moreover, results were obtained on a 600MHz instrument under protonated conditions. The goal of this project was the development of methodology to obtain structural data on a high-molecular weight protein associated with lipid nanodiscs in the presence of paramagnetic cofactors. Membrane proteins are challenging systems to research due to diverse interactions they experience on the membrane surface. In this dissertation I successfully utilized two approaches investigating this interactions: in my first project, I separately studied protein-protein interaction underlying the dimerization hypothesis, while in my second project I suggested the approach to explore conformational details and diverse interactions in a lipoprotein complex