The overall objective is to gain a more complete understanding of the structure of apolipoprotein (apo) E, especially as it relates to the ability of the protein to bind to heparin, the low density lipoprotein receptor (LDLR), and lipid particles of different size. Point mutations are known to give isoforms of apo E that function abnormally in cholesterol and triglyceride transport. A range of engineered apo E molecules expressed in E. coli is being used to address 3 specific aims. 1) To understand how interaction with phospholipid changes the conformation of apo E so that it can bind to the LDLR. The hypothesis being tested is that changes in alpha-helix organization alter the basic residue microenvironment in the LDLR binding domain (residues 136-150) which affects high affinity binding. NMR spectroscopy is used to monitor the microenvironments of lysine residues, and fluorescence and infrared spectroscopic methods are used to monitor the organization of apo E amphipathic alpha-helices in lipid-protein complexes. 2) To determine the type and organization of basic amino acids required in the apo E molecule for binding to heparin as opposed to the LDLR, and to understand why lipidation is required for binding of apo E to the LDLR but not for binding to heparin. The molecular mechanisms underlying changes in the energetics of binding arising from apo E polymorphism will also be investigated. 3) To understand the mechanisms responsible for the differing affinities of apo E isoforms for variously-sized serum lipoprotein particles, the molecular and thermodynamic parameters characterizing the binding of apo E and engineered variants to lipid particles of different sizes will be studied. The hypothesis being tested is that the length and properties (including the hydrophobicity of the nonpolar faces and the distribution of charged residues in the polar faces) of amphipathic alpha-helical segments in the C-terminal of the apo E molecule control lipid binding. Overall, achievement of these 3 aims will generate novel quantitative information about the ways in which apo E structure and polymorphism affect the functional properties of the protein in both physiological and pathological conditions. The design of ways to control the aberrant behavior of certain isoforms of apo E will be facilitated by this understanding.
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