The aim of this proposal is to test and apply a new technique (fluorescence lifetime heterogeneity analysis) for identifying the existence of microheterogeneity in membrane structure. The power of lifetime heterogeneity analysis is its potential sensitivity for detecting short-lived membrane microdomains which do not appear as thermodynamically distinct phases. The applicability of this technique will first be evaluated in simple model membrane systems in which structural heterogeneity is well established. Then, as initial applications, this method will be used to test two hypotheses stemming from work of the previous grant period: 1) that a trans-membrane protein both orders and disorders its local lipid environment, and 2) that cholesterol induces calorimetrically undetectable local domains in fluid lipid bilayers. New applications will use lifetime analysis to detect microstructures thought to be associated with two key cellular processes, membrane fusion and phospholipid trans-membrane migration. In addition to fluorescence lifetime analysis, appropriate thermodynamic techniques (scanning calorimetry, steady-state probe fluorescence, chemical labeling, freeze-fracture electron microscopy) will be used to examine bilayer structural features associated with these events in well-defined, model systems. In the case of polyethyleneglycol-induced membrane fusion, these measurements will test the hypothesis that different structural characteristics are associated with two crucial and previously inseparable intermediate states in the fusion process: bilayer close apposition and bilayer destabilization. In the case of cation-induced phosphatidylglycerol transbilayer redistribution, results will test two hypotheses: 1) transbilayer redistribution involves a two-component interaction between a metal ion and a lipid bilayer and 2) this interaction induces inverted micelle-like local structures in the lipid bilayer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032707-03
Application #
3281777
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1983-12-01
Project End
1986-11-30
Budget Start
1985-12-01
Budget End
1986-11-30
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Tarafdar, Pradip K; Chakraborty, Hirak; Bruno, Michael J et al. (2015) Phosphatidylserine-Dependent Catalysis of Stalk and Pore Formation by Synaptobrevin JMR-TMD Peptide. Biophys J 109:1863-72
Sengupta, Tanusree; Chakraborty, Hirak; Lentz, Barry R (2014) The transmembrane domain peptide of vesicular stomatitis virus promotes both intermediate and pore formation during PEG-mediated vesicle fusion. Biophys J 107:1318-26
Chakraborty, Hirak; Sengupta, Tanusree; Lentz, Barry R (2014) pH Alters PEG-mediated fusion of phosphatidylethanolamine-containing vesicles. Biophys J 107:1327-38
Chakraborty, Hirak; Tarafdar, Pradip K; Klapper, David G et al. (2013) Wild-type and mutant hemagglutinin fusion peptides alter bilayer structure as well as kinetics and activation thermodynamics of stalk and pore formation differently: mechanistic implications. Biophys J 105:2495-506
Tenchov, Boris G; MacDonald, Robert C; Lentz, Barry R (2013) Fusion peptides promote formation of bilayer cubic phases in lipid dispersions. An x-ray diffraction study. Biophys J 104:1029-37
Chakraborty, Hirak; Tarafdar, Pradip K; Lentz, Barry R (2013) A novel assay for detecting fusion pore formation: implications for the fusion mechanism. Biochemistry 52:8510-7
Majumder, Rinku; Koklic, Tilen; Rezaie, Alireza R et al. (2013) Phosphatidylserine-induced factor Xa dimerization and binding to factor Va are competing processes in solution. Biochemistry 52:143-51
Tarafdar, Pradip K; Chakraborty, Hirak; Dennison, S Moses et al. (2012) Phosphatidylserine inhibits and calcium promotes model membrane fusion. Biophys J 103:1880-9
Chakraborty, Hirak; Lentz, Barry R (2012) A simple method for correction of circular dichroism spectra obtained from membrane-containing samples. Biochemistry 51:1005-8
Majumder, Rinku; Liang, Xiaoe; Quinn-Allen, Mary Ann et al. (2011) Modulation of prothrombinase assembly and activity by phosphatidylethanolamine. J Biol Chem 286:35535-42

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