The plasma level of high density lipoprotein (HDL) is a primary indicator of clinical risk for the onset of atherosclerosis. This property is ascribed to the HDL's ability to facilitate reverse cholesterol transport (RCT), the major means of removing excess body cholesterol. As a principal facilitator of RCT, HDL initiates cholesterol removal from peripheral tissues via the ABCA1 transporter cholesterol efflux pathway. RCT plays a central role in lipid metabolism and cholesterol homeostasis by transporting lipids and cholesterol from peripheral tissues to the liver and steroidogenic tissues. Critical to RCT is the binding and sequestration of lipid by the primary protein component of HDL, apolipoproteinA-I (apoA-I), which, like other exchangeable apolipoproteins, associates with lipid by modulating the exposure of its hydrophobic residues through conformational adaptations. Despite its significance to RCT and lipid metabolism, the molecular details underlying apoA-I's lipid-induced conformational adaptation, and its regulation, are not well understood. To determine the molecular mechanisms governing this process, detailed structural knowledge of the manner in which apoA-I responds to changes in HDL geometry and size must be obtained. We will capitalize on our unique ability to purify the full repertoire of discoidal HDL subclasses. In this proposal, we will isolate 7.8, 8.4, 9.6 and 12.2 nm discoidal HDL and compare the structural parameters of apoA-I's intermolecular alignment and the presence of the apoA-I central loop, a domain critical for apoAI that our laboratory has recently characterized. Similarly, we will work in collaboration with Dr. Kerry Anne Rye who will transform our labeled and mutant apoA-I molecules used for these structural analyses to 9.7 nm spherical HDL. By comparing variations in apoA-I lipid-bound structures in these distinct HDL species, we will begin to understand the sequence of conformational transitions that apoA-I experiences as it adapts to a lipid environment and elucidate the functional consequences of these conformational changes. The proposed experiments not only provide insight into key functional domains of apoA-I, but place these domains into a structural context in which the wealth of data describing apoA-I's biochemical, metabolic, and physiological roles can be interpreted. This information will yield insight not only into apoA-I function but may provide a general mechanism by which lipoproteins report their lipid composition to their enzyme and receptor counterparts. PUBLIC HEALTH RELEVANCE Cardiovascular disease is one of the leading causes of mortality in first world nations and is a growing problem in developing countries. One of the primary indicators of increased risk of cardiovascular disease is a reduced level of HDL. This is attributed to HDL's anti-atherogenic character as a mediator of cholesterol transport. Apolipoprotein A-I (apoA-I) is the main protein component of HDL and is one of the primary facilitators of HDL function. As a result, therapies that target apoA-I and enhance its function in RCT are highly desirable. A greater understanding of apoA-I structure-function in HDL and RCT will aid us in formulating improved therapeutics based on apoA-I. Additionally, an enhanced understanding of apoA-I structure and HDL function will illuminate the cause of disease through dysfunction of HDL or apoA-I.