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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM048596-11
Application #
6619717
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Chin, Jean
Project Start
1993-01-01
Project End
2005-08-31
Budget Start
2003-08-01
Budget End
2005-08-31
Support Year
11
Fiscal Year
2003
Total Cost
$229,663
Indirect Cost
Name
State University New York Stony Brook
Department
Biochemistry
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Chiantia, Salvatore; Schwille, Petra; Klymchenko, Andrey S et al. (2011) Asymmetric GUVs prepared by M?CD-mediated lipid exchange: an FCS study. Biophys J 100:L1-3
Cheng, Hui-Ting; London, Erwin (2011) Preparation and properties of asymmetric large unilamellar vesicles: interleaflet coupling in asymmetric vesicles is dependent on temperature but not curvature. Biophys J 100:2671-8
Nelson, Lindsay D; Chiantia, Salvatore; London, Erwin (2010) Perfringolysin O association with ordered lipid domains: implications for transmembrane protein raft affinity. Biophys J 99:3255-63
Shahidullah, Khurshida; Krishnakumar, Shyam S; London, Erwin (2010) The effect of hydrophilic substitutions and anionic lipids upon the transverse positioning of the transmembrane helix of the ErbB2 (neu) protein incorporated into model membrane vesicles. J Mol Biol 396:209-20
LaRocca, Timothy J; Crowley, Jameson T; Cusack, Brian J et al. (2010) Cholesterol lipids of Borrelia burgdorferi form lipid rafts and are required for the bactericidal activity of a complement-independent antibody. Cell Host Microbe 8:331-42
London, Erwin; Shahidullah, Khurshida (2009) Transmembrane vs. non-transmembrane hydrophobic helix topography in model and natural membranes. Curr Opin Struct Biol 19:464-72
Cheng, Hui-Ting; Megha; London, Erwin (2009) Preparation and properties of asymmetric vesicles that mimic cell membranes: effect upon lipid raft formation and transmembrane helix orientation. J Biol Chem 284:6079-92
Zhao, Gang; London, Erwin (2009) Strong correlation between statistical transmembrane tendency and experimental hydrophobicity scales for identification of transmembrane helices. J Membr Biol 229:165-8
Shahidullah, Khurshida; London, Erwin (2008) Effect of lipid composition on the topography of membrane-associated hydrophobic helices: stabilization of transmembrane topography by anionic lipids. J Mol Biol 379:704-18
Nelson, Lindsay D; Johnson, Arthur E; London, Erwin (2008) How interaction of perfringolysin O with membranes is controlled by sterol structure, lipid structure, and physiological low pH: insights into the origin of perfringolysin O-lipid raft interaction. J Biol Chem 283:4632-42

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