Lipid-induced conformational switch of apolipophorin III

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DESCRIPTION (provided by applicant): Exchangeable apolipoproteins play a critical role in lipoprotein metabolism. They reversibly associate with lipoprotein surfaces, and are responsible for maintaining lipid homeostasis. Apolipoproteins play an important role in cardiovascular disease but details at the molecular level are still lacking. This project aims to investigate the molecular basis of lipid binding, and gain insight in the lipid-bound conformation of the apolipoprotein. This understanding would benefit the treatment and prevention of cardiovascular disease, in particular arteriosclerosis. The present study proposes to use a well characterized invertebrate apolipoprotein as a model, apolipophorin III (apoLp-III). A combination of structural analysis (circular dichroism and fluorescence spectroscopy) and functional analysis using model phospholipid membranes will be employed. The research plan contains the following aims: (i) investigate helix bundle stability as a function of lipid binding, (ii) identification of lipid factors that trigger apolipoprotein binding, and (iii) obtaining a high resolution structure of the protein in the lipid-bound form. (i) Helix bundle stability may provide a flexible protein facilitating helix bundle opening upon lipid binding. This opening is essential as it exposes the protein's hydrophobic interior to allow direct interaction with lipid. This will be tested by engineering mutant proteins with altered stability properties, and analysis of their lipid binding properties. (ii) Not much is known about the lipid factors that trigger apolipoprotein binding. Model bilayer vesicles composed of a variety of lipids will be used to gain insight in these lipid factors. (iii) The high resolution structure of lipid-free apolipoprotein is known, but still not known for the lipid-bound form. It is crucial to gain insight in this conformation as this is the biologically active form of the protein. We aim to obtain high quality crystals of apoLp-III/phospholipid complexes that diffract at approximately 5 angstrom. This would allow visualization of individual helices, thereby gaining insight in the helix arrangement of the apolipoprotein on the lipid surface.