This project involves exploiting the fluorescence quenching properties of nitroxide-labeled lipids, which when combined with other methods, allow the analysis of the details of membrane protein folding and membrane lipid organization. The hydrophobic helix is the basic structural unit of the vast majority of membrane proteins. To explore membrane protein folding, a systematic variation of the sequence of synthetic transmembrane helices will be employed, and their behavior assessed in lipid bilayers. In this system it is possible to introduce one or more amino acid residues (""""""""guest"""""""" residue) that may alter transmembrane insertion into any desired position(s) within the hydrophobic helix. In addition, lipid composition can be controlled. For each sequence, structure, membrane location, and helix-helix association will be determined, aided by a Trp residue included in each helix as a fluorescent probe. The effects of one, or more, """"""""guest"""""""" residue(s) upon helix properties will be assessed as a function of position within the hydrophobic core of the helix, or when positioned in the hydrophilic sequences flanking the core. The effect of altering the hydrophobic sequence into which the """"""""guest"""""""" residue is introduced, and lipid environment will also be studied. The knowledge gained will facilitate prediction of membrane protein tertiary structure, and thus both the design of membrane proteins de novo, and the ability to translate amino acid sequence data from genomic data into a knowledge of protein structure and function. The second goal is to use fluorescence quenching to study the organization of sphingolipid/cholesterol rich lipid domains (""""""""rafts"""""""") that have been proposed to play an important role in the regulation of variety of physiologically important functions within mammalian cell membranes, including signal transduction, protein sorting, viral budding, protein toxin action, amyloid formation, and prion maturation. Together with Dr. Deborah Brown the regulation of the formation of these domains by lipid and sterol composition (including biologically and medically important sterols) in vitro, and the effect of sterol structure on domain formation and function in cells, will be studied. How the structure of proteins, lipid anchors attached to proteins, and protein clustering affects the strength of protein association with domains will also be determined. Conversely the ability of proteins to promote domain formation will be evaluated.
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