The goal of this Project is to determine the structural motifs of apoB involved in assembly and subsequent three dimensional organization of apoB-containing lipoproteins. The proposed research is driven by four hypotheses:1) The N-terminal beta alpha1 domain of apoB forms a lipid pocket that initiates assembly of apoB-containing lipoproteins. 2) Binding of MTP to the alpha beta 1domain is required for formation of a functional lipid pocket. 3) The beta1 and beta 2 domains of apoB-100 form amphipathic beta sheets whose hydrophobic faces are in direct contact with the neutral lipid core of apoB-containing lipoproteins. 4) There is an increase in the number of amphipathic alpha helixes of the alpha 2 and alpha 3 domains associated with the surface lipids of the LDL particle as LDL size decreases, thus modulating the surface pressure decreases caused by a reduced radius of curvature. These hypotheses will be tested by four specific aims: 1) To determine the regions of the N-terminal domain of apoB that are required for the assembly of apoB-containing lipoproteins.
This aim will be accomplished by construction and expression of an extended library of C-terminally truncated forms of apoB, including those with site specific mutations, and analysis of the particles secreted by transfected mammalian cells in culture. 2) To study the functions of the amphipathic beta strands/sheets in apoB-100. A combination of chemical synthesis and molecular biology will be used to prepare apoB sequences containing variable numbers of amphipathic beta strands to compare their lipid-associating properties with amphipathic alpha helixes and to study the amphipathic beta strand to amphipathic beta transition on microemulsion surfaces. 3) To study the functions of the alpha 23 domains of apoB-100. ApoB sequences containing all or small regions of each of the domains will be expressed in E. coli, or short sequences chemically synthesized, and the resultant peptides studied., e.g., by monolayer techniques to determine surface exclusion pressure profiles. Model amphipathic peptides with defined surface pressures will also be used to determine the surface pressure of isolated LDL subclasses. 4). To develop a more detailed molecular model for LDL. A variety of molecular graphic methods will be used to build all-atom models into existing low resolution structures of LDL. One part of this aim will be to develop an all atom molecular model of the lipid pocket for apo B-containing lipoproteins.
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