| Control and regulation of membrane curvature play important roles in membrane trafficking and remodeling events. These processes are mediated by proteins that can sense and/or induce membrane curvature. The focus of my thesis is to understand the underlying molecular mechanisms that enable proteins to remodel membranes. Structural and biophysical studies were performed on the curvature-inducing proteins epsin (an ENTH protein) and endophilin (a BAR protein), both involved in membrane remodeling during endocytosis. Membrane interaction of alpha-synuclein, a curvature sensor, was also studied.;According to EPR and site-directed spin labeling, alpha-synuclein takes up an elongated helix with a helical periodicity of 11 amino acids per 3 turns (alpha11/3) in the membrane-bound form. Combining EPR and structural refinement, we found that the extended helical structure in the presence of membranes has a superhelical twist. This may be a result of alpha11/3 and allows the protein to have an elongated helical structure. The extended helix is at the level of the phosphate headgroup where it likely compensates for the curvature strain that is present in highly curved vesicles.;BAR domains are found among proteins involved in endocytosis and represents a membrane-binding and curvature-inducing module. The crystal structure of the BAR domain-containing protein endophilin show that it is a banana-shaped dimer composed of a 6-helical bundle arranged in a coiled-coil and suggests a scaffolding mechanism for the sensing and generation of membrane curvature. We found that many structural features of the crystal structure dimer are retained upon membrane interaction. The data also suggest that the BAR domain is at a distance from phosphate level of the membrane and is more likely to interact with the outermost region of the headgroup. The two regions not resolved in the crystal structure undergo a conformational change to a helical structure in the presence of membrane. The centers of both helices are at the level of the phosphate headgroup, where they are likely to promote membrane curvature by wedging lipids apart. Membrane-bound epsin was also analyzed.;Our work shows the importance of amphipathic helices. These studies will assist in the elucidation of the mechanism of membrane curvature regulation. |
