RATIONALLY DESIGNED ANALOGS OF AMPHIPATHIC HELIXES

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Abstract. The primary role of this project in the past, using peptide analogs of the amphipathic alpha helix, has been to experimentally test various hypotheses related to the structure and function of the amphipathic helical domains of apolipoproteins. The objective of the present project is to continue testing newly conceived hypotheses regarding structure-function relationships in apolipoproteins using more sophisticated experimental methods than used previously. This approach has already yielded new high-resolution structural information. Specific aims proposed are: (1) Development of rigorous computer algorithms to predict the structure of apolipoproteins associated with the surface of lipoprotein particles. (2) Studies of the determinants of lipid affinity and LCAT activation. (a) Depth of lipid penetration. The depth of lipid penetration of amphipathic helixes will be determined using fluorescence quenching and neutron diffraction. (b) Relative hydrophobicity of different amino acid residues. A hydrophobicity scale will be developed for each amino acid residue that is indexed to depth of lipid penetration. The approach will be to measure partition coefficients as the helix position and depth of penetration of a guest amino acid residue is varied within a host amphipathic alpha helix. ~ Orientation of the amphipathic helixes. Helix orientation in the discoidal complexes, lipid monolayers and supported lipid bilayers will be determined by polarized attenuated total reflectance fourier-transform infrared spectroscopy. (3) Structural studies of amphipathic alpha helixes in lipid or detergent complexes. (a) Two dimensional (2D) 1H- NHR studies of lipid complexes of amphipathic peptides. Based upon the exciting preliminary results obtained, we propose to initiate the structural studies of the amphipathic peptides in the presence of lipid using 2D 1H-NMR spectroscopy. (b) X-ray crystallography. In collaboration with Dr. Michael Garavito of Michigan State University, we propose to crystallize peptide analogs of amphipathic alpha helixes in the presence of detergents and/or lipids.