Research

Caveolin

The primary concern of our research is the investigation and characterization of membrane proteins and their interactions with the lipid bilayer. Of particular interest to our research group is caveolin-1, a membrane protein that is pivotal in the formation of plasma membrane invaginations known as caveolae (pictured left).

Caveolae are involved in a wide variety of essential cellular functions pertaining to the plasma membrane, and their misregulation has been implicated in cancers, Alzheimer's disease, and many other human diseases. While the role of caveolae in cell biology has been well established, the exact role that caveolin-1 plays in the formation and homeostasis of caveolae on a fundamental level remains unclear.

Our research focuses on taking a biochemical and biophysical approach to understanding the structure, oligomerization, and topology of the caveolin protein.

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Structure

Caveolin-1 is a very interesting protein from a structural perspective. The hallmark feature of the protein is a putative intra-membrane loop or V-shaped configuration. Using protein NMR techniques, we have determined the secondary structure of a functional construct of caveolin-1 in detergent micelles. The studies revealed the presence of three helices (1, 2, 3), and is consistent with the proposed structure of the protein. Helices 1 & 2 form the intra-membrane loop (V-shape) with the turn occurring at the residues glycine, isoleucine, and proline (GIP). Helix 3 forms an amphipathic helix which rest horizontally on the surface of the bilayer. Current and future studies include probing the role of palmitoylation on the secondary structure of caveolin-1 as well as structure detemination in a more native like membrane mimic such as bicelles or nanodiscs.

Topology

Caveolin-1 is proposed to have an unusual topology where both the N- and C- termini of the polypeptide chain reside in the cytosol, and the central portion of the protein forms a membrane-embedded hairpin or loop type structure. Creation of single tryptophan mutants has allowed us to probe the position of each tryptophan with respect to the bilayer. Tryptophan is a naturally fluorescent amino acid, and the emission maximum depends on the polarity of the medium in which the tryptophan resides. For example, a tryptophan which is exposed to water has an emission maximum of ~350 nm, while a tryptophan residue in the center of a lipid bilayer has an emission maximum of ~333 nm. Our data are consistent with the proposed hairpin topology of caveolin as two of the tryptophans are buried in the hydrophobic core of the bilayer while the other two are located in the headgroup region of the bilayer. Current and future studies include probing the location of other amino acid positions in caveolin-1 to form a more definitive picture of its topology.

Oligomerization

Caveolin-1 is a monomer in detergent micelles. Proline 132 in the amino acid sequence of caveolin-1 plays an important role in supressing dimerization. Mutation of proline 132 to other amino acids (i.e., leucine, isoluecine, glycine, alanine, phenylalanine, valine, and tryptophan) triggers stable dimerization formation. The figure to your left is data from the analytical ultracentrifuge. The wild type data fits well to a monomer model, while the proline 132 to leucine data fits to a dimer model. Current and future studies involve investigating the oligomeric state of caveolin-1 in native membranes with and without cholesterol.

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