Our group has developed a spectroscopic method allowing measurement of the transverse location (depth) of sites in a membrane at the angstrom level of resolution. It involves fluorescence quenching measurements using phospholipids carrying nitroxide quenchers at different depths. Simple algebraic expressions are then used to calculate depth. Procedures have now been developed which allow accurate measurements over wide range of depths. In the next project period, the method will be used to investigate a fundamental issue of membrane structure, namely, the rules governing the relationship of the chemical structure of membrane-interacting molecules to their depth. As part of this aim depth will be measured for s systematic series of hydrophobic and lipid-linked probes we will prepare such that the attachment site, probe polarity, and the nature of the reactive group on the probe is varied. The effect of cholesterol on depth will also be determined. This is of particular interest in view of recent studies showing that cholesterol and sphingolipid-rich domains may exist in cellular membranes. In collaborative studies, the regulation of the formation of such domains may exit in cellular membranes. In collaborative studies, the regulation of the formation of such domains by lipid composition, and their interactions with proteins will be examined. The quenching method will also be used to determine how amino acid composition regulates the structure of membrane inserted polypeptides. This will be examined by depth measurements on derivatives of known transmembrane inserted polypetides. This will be examined by depth measurements on derivatives of known transmembrane a-helical hydrophobic peptides into which different types and numbers of polar residues have been introduced at specific positions. Measurement of the depth of a single Trp residue in these peptides, in conjunction with circular dichroism and energy transfer measurements, will allow facile analysis of the effects of the substitutions on peptide structure, and the formulation of rules relating transmembrance orientation to sequence. The method will also be applied to the high resolution topography of membrane proteins. This will be done by measuring the depth of single fluorescent groups introduce at a series of residues by site-directed mutagenesis. First, the calibration of the method and identification of the best fluorescent labels for such studies will be completed by a fuller comparison of the theoretical and experimental depths for single site-labeled transmembrane peptides. Then, the method will be applied to a membrane protein for which a series of single Cys mutants are now available, the a-hemolysin of S. Aureus.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM048596-07
Application #
6018933
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1993-01-01
Project End
2000-07-31
Budget Start
1999-08-01
Budget End
2000-07-31
Support Year
7
Fiscal Year
1999
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Biochemistry
Type
Schools of Arts and Sciences
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
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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
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
London, Erwin; Shahidullah, Khurshida (2009) Transmembrane vs. non-transmembrane hydrophobic helix topography in model and natural membranes. Curr Opin Struct Biol 19:464-72
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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