Membrane binding kinetics and curvature partitioning of peripheral proteins

The localization of peripheral proteins to specific sites of cell membranes is a pivotal step in many cellular processes. Many such processes involve dynamic changes in membrane structure and particularly membrane curvature. Although certain proteins have been implicated in these processes, many questions remain as to the mechanisms underlying their involvement. A major goal toward this end is to elucidate mechanisms by which they generate and sense membrane curvature. The membrane curvature sensing and generation properties of the peripherally binding endocytic accessory protein endophilin are believed to depend on its dimeric structure. We therefore first study endophilin dimerization kinetics and equilibria in solution, employing Förster resonance energy transfer techniques. Mechanistic insights into the association of endophilin N-BAR with membranes are obtained from kinetic studies by stopped-flow rapid mixing techniques. We incorporate our findings into a kinetic scheme for endophilin N-BAR membrane binding that highlights contributions from membrane-mediated protein oligomerization, and obtain kinetic parameters of the model through comparison to experimental data. Secondly, we investigate the partitioning of peripheral proteins between connected membrane regions of differing curvature using microscopy and micromanipulation methods, particularly focusing on epsin and amphiphysin. This dissertation thus sheds light on multiple properties of peripheral binding proteins to elucidate the basis for their involvement in membrane trafficking processes: solution assembly state, kinetics of membrane association, oligomerization on the membrane, and curvature partitioning. The implications of each of these factors for the regulation of membrane trafficking processes involving the proteins studied are discussed