Biophysical Studies of Caveolin - Project Summary/Abstract Caveolae are flask-shaped invaginated microdomains that punctuate the plasma membrane, and play a central role in a variety of cellular processes including mechanosensing, endocytosis, and signal transduction. The integral membrane protein caveolin (three isoforms -1, -2, and -3) is the most important protein found in caveolae, and is required for caveolae biogenesis. A plethora of studies have shown that improper regulation and mutant forms of caveolin can result in a variety of diseases including lipodystrophy, muscular dystrophy, cancer, and heart and lung disease. Based on indirect evidence, the provocative postulation has been put forth that caveolin adopts a ‘U-shaped’ conformation in the lipid bilayer, a disposition that to our knowledge has not been definitively characterized for any membrane protein. Furthermore, evidence suggests that caveolin has the ability to homooligomerize, although recent evidence from our lab has challenged that notion and brought into focus the uncomfortable possibility that many of the methods used to study caveolin may be inadvertently promoting non-biologically relevant aggregation. Using a panoply of biophysical and biochemical techniques (i.e. fluorescence spectroscopy, circular dichroism spectroscopy, nuclear magnetic resonance [NMR] spectroscopy, chemical cross-linking, and computational modeling) our objective is to probe the structure, topography and homooligomeric state of caveolin-1 (the most ubiquitous of the three isoforms) in a lipid bilayer. This will be achieved by pursuing the following three specific aims. 1. Investigation of the putative ‘U-shaped’ conformation of the intramembrane domain. 2. Investigation of the secondary structure of the N-terminal domain. 3. Investigation of homooligomeric interactions. Specific aim 1 will determine the tertiary structure and membrane topography of the intramembrane domain which will address the persistent skepticism surrounding the atomic-level interactions that could make a membrane-buried polypeptide turn possible. Specific aim 2 will address the long standing question of whether any 𝛂-helices or β-strands are present in the N-terminal domain, and with the fulfillment of this aim, complete backbone NMR data will finally be available for caveolin-1. Specific aim 3 will evaluate whether caveolin-1 possesses the ability to oligomerize on its own when reconstituted into the bilayer at in vivo (i.e. high) surface densities by devising an experiment that will circumvent the pitfalls that may have derailed previous investigations into oligomerization and led to false conclusions (e.g. use of detergents, tagging of the protein, etc.). Upon completion of these aims, the key enigmatic features of caveolin-1 will be manifest, opening the door to deeper insights into disease pathogenesis and ultimately possible therapeutic interventions that could address the immensely complex array of disease states linked to aberrant caveolin function.
