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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062305-02
Application #
6490162
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Chin, Jean
Project Start
2001-01-01
Project End
2004-12-31
Budget Start
2002-01-01
Budget End
2002-12-31
Support Year
2
Fiscal Year
2002
Total Cost
$175,864
Indirect Cost
Name
University of Virginia
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Lai, Alex L; Huang, Hao; Herrick, Dawn Z et al. (2011) Synaptotagmin 1 and SNAREs form a complex that is structurally heterogeneous. J Mol Biol 405:696-706
Kuo, Weiwei; Herrick, Dawn Z; Ellena, Jeffrey F et al. (2009) The calcium-dependent and calcium-independent membrane binding of synaptotagmin 1: two modes of C2B binding. J Mol Biol 387:284-94
Bhowmik, Anshu; Ellena, Jeffrey F; Bryant, Robert G et al. (2008) Spin-diffusion couples proton relaxation rates for proteins in exchange with a membrane interface. J Magn Reson 194:283-8
Huang, Hao; Cafiso, David S (2008) Conformation and membrane position of the region linking the two C2 domains in synaptotagmin 1 by site-directed spin labeling. Biochemistry 47:12380-8
Liao, Haini; Ellena, Jeff; Liu, Lixia et al. (2007) Secretory carrier membrane protein SCAMP2 and phosphatidylinositol 4,5-bisphosphate interactions in the regulation of dense core vesicle exocytosis. Biochemistry 46:10909-20
Fanucci, Gail E; Cafiso, David S (2006) Recent advances and applications of site-directed spin labeling. Curr Opin Struct Biol 16:644-53
Rufener, Elisabeth; Frazier, April A; Wieser, Catherine M et al. (2005) Membrane-bound orientation and position of the synaptotagmin C2B domain determined by site-directed spin labeling. Biochemistry 44:18-28
Ellena, Jeffrey F; Moulthrop, Jason; Wu, Jing et al. (2004) Membrane position of a basic aromatic peptide that sequesters phosphatidylinositol 4,5 bisphosphate determined by site-directed spin labeling and high-resolution NMR. Biophys J 87:3221-33
Gambhir, Alok; Hangyas-Mihalyne, Gyongyi; Zaitseva, Irina et al. (2004) Electrostatic sequestration of PIP2 on phospholipid membranes by basic/aromatic regions of proteins. Biophys J 86:2188-207
Frazier, April A; Roller, Christina R; Havelka, Jessica J et al. (2003) Membrane-bound orientation and position of the synaptotagmin I C2A domain by site-directed spin labeling. Biochemistry 42:96-105

Showing the most recent 10 out of 14 publications