Despite enormous recent progress in structural biology in general, determining the structure of membrane proteins has remained difficult. Similarly, we understand very little about the mechanism by which proteins interact with membranes. Here I propose to study this process using site-directed spin labeling (SDSL). SDSL has become a powerful new technique for determining structure and conformational dynamics in soluble and membrane proteins. SDSL is not limited to a particular protein size and can be used to monitor conformational changes in real time under physiological conditions. The primary model system for these studies will be annexin 12, a member of the annexin family of membrane binding proteins. Annexin 12 has three distinctly different states: (1) a water-soluble state of known structure, (2) a Ca2tdependent, peripherally membrane-bound form, and as we recently discovered, (3) a transmembrane form that is lipid and pH dependent, but does not require Ca2+ It is the goal of this proposal to investigate the structures of the peripheral and integral membrane-associated states and determine the factors that modulate the reversible interconversion between them. In addition, we expect this structural work to provide an important foundation to evaluate and rationalize the numerous membrane-related functions of annexins. Sequence analysis suggests that other proteins also could insert into membranes using an annexin-like mechanism. Because of intriguing physiological implications, we will study the membrane interaction of one of these proteins, the estrogen receptor alpha.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-SSS-B (01))
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Shapiro, Bert I
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University of Southern California
Schools of Medicine
Los Angeles
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Marotta, Nicholas P; Lin, Yu Hsuan; Lewis, Yuka E et al. (2015) O-GlcNAc modification blocks the aggregation and toxicity of the protein ?-synuclein associated with Parkinson's disease. Nat Chem 7:913-20
Ambroso, Mark R; Haworth, Ian S; Langen, Ralf (2015) Structural Characterization of Membrane-Curving Proteins: Site-Directed Spin Labeling, EPR, and Computational Refinement. Methods Enzymol 564:259-88
Kegulian, Natalie C; Sankhagowit, Shalene; Apostolidou, Melania et al. (2015) Membrane Curvature-sensing and Curvature-inducing Activity of Islet Amyloid Polypeptide and Its Implications for Membrane Disruption. J Biol Chem 290:25782-93
Isas, J Mario; Ambroso, Mark R; Hegde, Prabhavati B et al. (2015) Tubulation by amphiphysin requires concentration-dependent switching from wedging to scaffolding. Structure 23:873-881
Lai, Ying; Kim, Sunae; Varkey, Jobin et al. (2014) Nonaggregated ?-synuclein influences SNARE-dependent vesicle docking via membrane binding. Biochemistry 53:3889-96
Ambroso, Mark R; Hegde, Balachandra G; Langen, Ralf (2014) Endophilin A1 induces different membrane shapes using a conformational switch that is regulated by phosphorylation. Proc Natl Acad Sci U S A 111:6982-7
Shah, Claudio; Hegde, Balachandra G; Morén, Björn et al. (2014) Structural insights into membrane interaction and caveolar targeting of dynamin-like EHD2. Structure 22:409-420
Cheng, Chi-Yuan; Varkey, Jobin; Ambroso, Mark R et al. (2013) Hydration dynamics as an intrinsic ruler for refining protein structure at lipid membrane interfaces. Proc Natl Acad Sci U S A 110:16838-43
Moaven, Hormoz; Koike, Yukihiro; Jao, Christine C et al. (2013) Visual arrestin interaction with clathrin adaptor AP-2 regulates photoreceptor survival in the vertebrate retina. Proc Natl Acad Sci U S A 110:9463-8
Ruggiero, Linda; Connor, Mark P; Chen, Jeannie et al. (2012) Diurnal, localized exposure of phosphatidylserine by rod outer segment tips in wild-type but not Itgb5-/- or Mfge8-/- mouse retina. Proc Natl Acad Sci U S A 109:8145-8

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