The long terms goal of this research is to elucidate the metabolic significance of exchangeable apolipoprotein structural alterations. Studies will focus on a key member of this protein class, human apolipoprotein E (apoE). While structural information is available for this protein in the absence of lipid, it is recognized that exchangeable apolipoproteins exert their biological effects only in a lipid-associated state. Evidence suggests these proteins undergo significant conformational changes) upon lipid binding. The N-terminal domain of apoE is organized as a bundle of elongated amphipathic alpha-helices. Models have been proposed which predict the helix bundle can open about a putative hinge domain located in the loop between helices. Such a conformational change would result in exposure of hydrophobic residues, making them available for interaction with lipoprotein surfaces. It is proposed that this conformational change is reversible and that helix boundaries present in the lipid-free conformation are maintained in the lipid associated state. The precise nature of lipid binding-induced conformational adaptations of apoE N-terminal domain will be determined. Structural information will be used to select candidate amino acid residues for site directed mutagenesis. Disulfide bond engineering and fluorescence resonance energy transfer will be performed to evaluate lipid binding-induced helix repositioning. Mutant proteins containing a single tryptophan and a single cysteine will be expressed in bacteria. Modification of cysteine with an appropriate chromophore provides an energy acceptor from excited tryptophan for distance measurements. Through judicious placement of energy donor/acceptor pairs in the molecule, it will be possible to construct a map of helix movements upon lipid binding. It is hypothesized that conformational opening of the N-terminal domain of human apoE represents a physiologically important mechanism for regulation of its receptor binding activity. Studies will be performed to characterize the correlation between lipid-associated full length apoE interactions with the low density lipoprotein receptor on cultured human skin fibroblasts and the conformational status of its N-terminal domain. The results obtained will provide new information about the physiological relevance of the conformational adaptability of exchangeable apolipoproteins.
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