Many proteins that are involved in cell signaling pathways are water-soluble but participate in signaling only when they are associated with the membrane interface. The overall objective of this project is to determine the structures and forces that govern the association of proteins to membrane interfaces. Protein domains known as C2 domains are the most prevalent Ca2+ signaling motif found in eukaryotic systems and they function to attach proteins to the membrane-solution interface in a Ca2+-dependent fashion. These domains play critical roles in processes such as the release of lipid-derived second messengers, protein phosphorylation, membrane trafficking, protein ubiquitination, membrane pore formation, and GTPase regulation. Although high-resolution solution and crystal structures for these domains are known, information on how these domains are positioned on the membrane and interact with the lipid interface is limited. The proposed work will determine the orientation and position of C2 domains from cPLA2 and synaptotagmin on the membrane interface by the use of site-directed spin-labeling and EPR spectroscopy. This methodology will also be used to investigate structural changes that accompany Ca2+ and membrane binding, and a novel approach to determine electrostatic potentials will be used to estimate potentials at the lipid binding surfaces of C2 domains. This information will be utilized to evaluate the mechanisms of membrane attachment of these domains. Another mechanism for the membrane attachment of proteins involves the electrostatic interaction of basic protein motifs with acidic lipid interfaces. Such interactions are commonly found in proteins involved in cell-signaling and they account for the association of protein kinases such as src, and regulatory proteins such as MARCKS. The proposed work will investigate the positions of these domains within the electrostatic double layer and evaluate the forces acting on these domains that attach them to the membrane interface. We anticipate that a better understanding of the forces and mechanisms that attach proteins to the membrane interface will facilitate a better understanding the of the regulation and timing of cell-signaling events. A better understanding of these interactions may also lead to the development of new approaches to control cell-signaling and cell growth.
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