Investigation of the Three-Dimensional Structure of the Caveolin-1 membrane Interacting Domain

Abstract

The importance of understanding membrane protein structure and function is growing after scientists have recognized that approximately one third of the genome encodes for membrane proteins. Caveolin is an integral membrane protein that is found in high abundance in caveolae. Caveolae are invaginations of the plasma membrane that play an important role in many cellular functions. Interestingly, caveolin plays a dual-role in caveolae. On one hand it is responsible for forming the “flask-like” shape of caveolae, and on the other hand it is intimately involved in cellular processes such as signal transduction and protein trafficking. Alterations in the levels of caveolin protein and mutations of the caveolin protein have been implicated in a variety of disease states including cancer, heart disease, and muscular dystrophy. Caveolin possesses a unique hairpin-like topology (N- and C- termini are on the same side of the membrane) in the membrane which is thought to facilitate its functions. In particular, mutations of two proline residues (P110A and P132L) in the caveolin-1 transmembrane domain appear to be critical for proper biological function, and serve as markers for various disease states. To study the effects of these important mutations, we have prepared constructs of the caveolin protein that encompass the transmembrane domain. Both Caveolin(99-137) and caveolin(122-142) were over-expressed and isotopically labeled in E.coli, purified to homogeneity, and incorporated into dodecylphosphocholine micelles. The three-dimensional structure of the constructs will be revealed using a combination of circular dichroism and NMR spectroscopy. Moreover, comparison of the wild-type structure with that of the constructs containing the proline mutations will give valuable insight into the unusual nature of the caveolin-1 transmembrane domain.