Investigation of Caveolin Structure, Topology, and Oligomerization

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DESCRIPTION (provided by applicant): Caveolae are highly-curved invaginated micro-domains located in the plasma membrane that play a central role in a variety of cellular processes. Caveolins (1, 2, and 3) are the most important proteins found in caveolae, and are responsible for giving caveolae their unusual "flask-like" shape. Recent evidence has shown that improper regulation and mutant forms of caveolin can result in a variety of diseases including Alzheimer's, muscular dystrophy, cancer, and heart disease. Caveolin adopts an unusual intra-membrane "horseshoe" conformation where both its N- and C-termini face the cytoplasm, and this conformation is thought to promote membrane curvature. In addition, via high-order oligomerization, caveolin forms a structural backbone which stabilizes the membrane curvature. Using biophysical techniques such as nuclear magnetic resonance (NMR), fluorescence spectroscopy, and analytical ultracentrifugation, our objective is to characterize caveolin-1 on a fundamental level. This will be achieved by pursuing the following two specific aims: 1. Investigation of the membrane topology and three-dimensional structure of caveolin-1. 2. Investigation of caveolin-1 oligomerization. Specific aim 1 will determine the high-resolution three-dimensional solution structure of caveolin-1 as well as examine the solvent accessibility of tryptophan residues to assess the topology of caveolin-1 in a bilayer. Next, the role that two conserved proline residues play in the creation and/or stabilization of the intra-membrane "horseshoe" conformation will be probed using site-directed mutagenesis. Specific aim 2 will characterize both the size and distribution of oligomers formed by caveolin-1 in the presence and absence of cholesterol. Additionally, the role that a proline to leucine mutant plays in the oligomerization process will be probed. A fundamental understanding of caveolin-1 structure and oligomerization will undoubtedly open the door to possible therapeutic interventions that could address diseases linked to caveolin misfunction.