The goal of this proposal is to understand the structural basis of apolipoprotein (apo) A-I function. The proposed research is based upon a detailed molecular double belt model we have developed for the discoidal form of high density lipoprotein (HDL) and is driven by four hypotheses: 1) Interhelical salt bridges between the two belts are involved in one or more rotationally regulated biological functions. 2) Folding variations in apoA-I on the disc edge have functional implications. 3) Basic residues on the outside edges of the double belt facing the head groups of the disc have specific functions involving disc stability and interactions with enzymes and other apolipoproteins. 4) The detailed model will lead to development of additional models relating to HDL structure-function, including the role of the N-terminal 43 residues of apoA-I and the conformation of apoA-I on spheroidal HDL. These hypotheses will be tested by five specific aims: 1) To determine the prevalence and function of putative interhelical salt bridges.
This aim will be accomplished by construction, expression and biophysical/functional studies of carefully designed mutants of wt apoA-I, and certain truncated forms, in which rotamers are fixed by cross-linking or putative salt-bridges are added or subtracted. 2) To develop new models of apoA-I:lipid complexes. A combination of molecular dynamics, electrostatic potential and molecular graphic approaches will be used. 3) To determine the prevalence and function of the amphipathic alpha helical double belt versus the hairpin-belt conformers. Site-directed mutagenesis will be used to add cysteines at key positions to produce disulfide cross-linking of lipid-associated dimers of apoA-I in a manner to distinguish between the two conformations. The effects of disc size, phospholipid and cholesterol composition and method of disc formation on apoA-I conformation will be determined. 4) To determine the function of the N-terminal 43 residues of apoA-I. This domain will be modified by sequential deletion of the three tandem 11-mer domains and the properties of the mutants examined. 5) To determine the function of the basic residues on the outside of the amphipathic alpha helixes that face the head groups of the disc. Site-directed mutagenesis will used to add or delete basic residues in this position and the effects on apoA-I properties examined.
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