Prioritized project goals are to: (a) test the hypothesis that the three amphipathic helical domains of apo B ( alpha 1, alpha 2, and alpha 3) act as independent structural and functional units; (b) investigate the role of amphipathic beta strands and/or antiparallel amphipathic beta sheets in the interactions of apo B with lipoprotein surfaces, and ~ in a more risky sub-project, explore the structural features of apo B that are involved in assembly of apo B-containing lipoproteins. The research strategy involves studies of the biophysical and biological properties of: 1) rationally designed apolipoprotein B fragments, created and expressed in vitro by cultured cells and in intact lipoproteins by cells in culture by Core D, and 2) shorter sequences produced by chemical peptide synthesis by Core B. WHEEL and LOCATE computer algorithms (Project 1, in collaboration with Project 2) will be used to design mutations. To achieve the above goals, two specific aims are proposed: 1) Studies of rationally designed site- directed mutants of apo B. (a) We will test the hypothesis that the three amphipathic helical domains of apo B (alpha 1, alpha 2, and alpha 3) act as independent structural and functional units and that the alpha 1 domain is ~globular~ and can be crystallized. Each domain will be expressed in cells in culture and studied. (b) We will test the hypothesis that amphipathic beta strands from the N- terminal 18-28% of apo B, in association with the alpha 1 domain, form a lipovitellin-like intermediate that contains a lipid pocket required to initiate the assembly of triglyceride-rich lipoprotein particles. ~ An additional hypothesis to be tested is that this lipovitellin-like intermediate structure is not complete without structural integration of MTP.2) Studies of lipid interactions of amphipathic beta strand and sheets. In addition to amphipathic helixes, putative amphipathic beta strands have been located in the primary structure of apo B. We propose to study the structure and lipid associating properties of native (from apo B-100) as well as de novo designed amphipathic beta strands and sheets. Both peptide synthesis and molecular biology techniques will be used to obtain these peptides.
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