The reversible binding of proteins to intracellular membrane interfaces is critical to the regulation of membrane trafficking and cell-signaling pathways. This association activates enzymes, facilitates protein- protein interactions, drives the lateral organization of protein assemblies and is responsible for the remodeling of cell membranes. It has also been shown to regulate cell-transformation and cancer. Membrane binding is often mediated by specialized protein domains that associate with the membrane interface in response to specific cellular signals;however, there is little information about the membrane interactions made by these domains. The proposed work will use an EPR based technique termed site- directed spin labeling to determine the membrane orientation and position of these domains. The forces that drive membrane attachment will also be evaluated. The C2 domains of synaptotagmin mediate calcium- dependent neuronal exocytosis, and their positions on the membrane interface in the presence of phosphatidylinositol-4,5-bisphosphate (an important signaling lipid) will be determined. Electrostatic forces may drive the assembly of signaling complexes, and the hypothesis that highly positively charged protein segments act as a scaffold to sequester proteins on the bilayer surface will be tested. The membrane position of two phosphoinositide binding domains will also be characterized and new approaches to determine protein orientation and depth on membrane surfaces evaluated. We anticipate that a better understanding of these protein-membrane interactions will lead to a better understanding of membrane trafficking and cell-signaling events. A better understanding of these events may also lead to the development of new approaches to control cell growth and cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062305-08
Application #
7535504
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
2001-01-01
Project End
2010-11-30
Budget Start
2008-12-01
Budget End
2010-11-30
Support Year
8
Fiscal Year
2009
Total Cost
$213,276
Indirect Cost
Name
University of Virginia
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
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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

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