Our group has recently developed a novel fluorescence method that allows the measurement of the transverse location (depth) of specific sites within a membrane relative at the angstrom level of resolution. The approach, which we have named the parallax analysis, involves fluorescence quenching measurements using phospholipids carrying spin- labeled quenchers. We derived simple algebraic expressions that allow calculation of fluorophore depth from such experiments.
The aim of this project is to further develop and apply the method to solve questions of membrane structure. Specifically: 1) The method will be applied to a wide range of membrane probes to determine the chemical/structural factors that control depth of molecules in membranes. By correlation of depth with polarity, charge, number of heteroatoms, hydrogen bonding, and other structural features it should be possible to identify which properties influence depth most strongly. 2) The applicability of the method will be extended to the extremities of the polar headgroup and adjacent aqueous solution by the use of a spin-label attached to the polar headgroup of a lipid. Another goal is to extend the method to distinguish which half of a bilayer a group is found in, an important parameter for membrane-inserted proteins. this will be done by using ascorbate reduction of spin-labels in half of the membrane to restrict quenching to a single leaflet of the bilayer. 3) In collaborative studies the method will be used to analyze the topography of membrane proteins. To accomplish this the location of single fluorescent Trp residues substituted into the alpha-helical membrane protein lactose permease will be determined. Similar studies will be undertaken with the pore-forming beta sheet alpha-toxin of S. aureus. In this case, single fluorescent sites will be created by labeling of mutants containing single Cys residues. Together these studies should establish the method as a practical approach to questions of membrane structure and function.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM048596-03
Application #
2186093
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1993-01-01
Project End
1996-12-31
Budget Start
1995-01-01
Budget End
1995-12-31
Support Year
3
Fiscal Year
1995
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
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
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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