Apolipoprotein A-I (apoAI) is a 243-residue exchangeable apolipoprotein that plays a key role in clearing the """"""""bad cholesterol"""""""" from the human blood stream. Depending on the extent of lipidation, apoAI may adopt one of four distinct conformations, including: (1). Lipid-free apoAI conformation (Specific phospholipid-binding). (2). ApoAI on pre-beta-HDL (Specific cholesterol binding). (3). ApoAI on discoidal HDL (Specific LCAT activation). (4). ApoAI on spherical HDL (Specific SR-BI, the HDL-receptor, binding). The conformational plasticity indeed enables apoAI to perform multiple functions which regulate/direct HDL formation, maturation, transport and metabolism. Currently we do not have a clear understanding of how the apoAI structural domains allow for this structural plasticity. This proposal concentrates on determining the NMR structures/dynamics of the lipid-free and pre-beta-HDL-bound apoAl. Based on preliminary data and published results by other laboratories, the following hypotheses are proposed: (1). Lipid-free apoAI mainly adopts a helix-bundle structure. (2). Phospholipid-binding induces a dramatic conformational change of apoAI that exposes the hydrophobic sites for cholesterol binding. (3). ApoAI/DPC mimics the structure and functions of apoAI/pre-beta-HDL, thus may have therapeutical implications to atherosclerosis. In order to verify these hypotheses, we propose to solve the NMR structures of apoAI in three different states: (1). Lipid-free state, (2). ApoAI/DPC state, (3). ApoAI/pre-beta-HDL state. Functional characterizations of apoAI/DPC and apoAI/pre-beta-HDL will also be performed, in terms of cholesterol-binding activity. In addition, NMR techniques will be utilized to study structural dynamic of apoAI in these three states. These studies together may allow us to address the structural switching mechanism, which converts apoAl's conformation from one to another. It is anticipated that the structures of lipid-free and pre-beta-HDL bound apoAI will help in the understanding of how apoAI recruits lipid to initiate the HDL formation, and how apoAI promotes pre-beta-HDL to recruit more neutral lipids for the maturation of HDL. Since a low level of plasma HDL and a compromised HDL function are the common thread of metabolic disorders/diseases including: atherosclerosis, diabetes, obesity, stroke, and Alzheimer's disease, the results obtained from this proposal should have significant implications for the intervention of new medicine to treat these metabolic disorders/diseases.
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