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NMR structural studies of apoAI/preb-HDL particles


EMSL Project ID
3999

Abstract

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. ApoAI is the most abundant protein component in high-density lipoprotein (HDL, ~70%). Depending on the extent of lipidation, apoAI displays a dramatic conformational plasticity and may adopt one of four distinct conformations, including: (1). Lipid-free apoAI conformation (for specific phospholipid-binding, M.W.: 28 kDa). (2). ApoAI on preb-HDL (for specific cholesterol-binding, M.W.: ~50 kDa). (3). ApoAI on discoidal HDL (for specific LCAT activation, M.W.: ~140-160 kDa). (4). ApoAI on spherical HDL (for specific HDL-receptor binding, M.W.: 12-180 kDa). Preb-HDL has been known as the first acceptor and the most active acceptor of free cholesterol. It is the apoAI protein on preb-HDL that displays a unique conformation with the exposed binding sites specifically for free cholesterol. Thus, the apoAI conformation on preb-HDL represents one important conformation of this protein that triggers the HDL formation. The low metastability of preb-HDL has made a structural/functional characterization of this particle difficult. Thus, there have been no structural studies of the apoAI/preb-HDL particles published to date. Preb-HDL particles contain only one apoAI molecule with a particle size of ~6.0 nm, thus it is certainly workable for NMR structural studies using the newly developed TROSY NMR techniques. In collaboration with Dr. Dan Sparks at the University of Ottawa, we prepared stable preb-HDL which is stable for weeks at ~10 mg/ml of apoAI concentration. We further showed that high-quality NMR data has been collected which could be used for a NMR structural determination of apoAI on preb-HDL. This proposal concentrates on NMR structural studies of apoAI/preb-HDL particles, and plan to collect 3D/4D-NMR experiments at high-field, such as 750/800 MHz, for an NMR spectral assignment and structural calculation of apoAI/preb-HDL.

Project Details

Project type
Capability Research
Start Date
2003-10-01
End Date
2004-10-11
Status
Closed

Team

Principal Investigator

Jianjun Wang
Institution
Wayne State University

Team Members

So-Young Shin
Institution
Wayne State University

Jianglei Chen
Institution
Wayne State University

Bin Chen
Institution
Wayne State University