Poly(ethylene glycol) (PEG) is used to mediate cell-cell fusion in the production of somatic cell hybrids and in the fusion injection of macromolecules into cultured cells from erythrocytes or liposomes. In the previous funding period, we defined in greater molecular detail the mechanism of PEG-mediated fusion and showed that it is remarkably similar in kinetically to the kinetic behaviors defined by electrophysiological and fluorescence studies of influenza virus hemagglutinin-mediated fusion of cell membranes and fusion of secretory vesicles with secretory cell surface membranes. This has allowed us to pursue the mechanism of PEG-mediated fusion as a mimetic for much more complex protein-mediated cell membrane fusion. We have determined the optimal lipid composition for PEG-mediated fusion and found this to be nearly the same as the composition of synaptic vesicle membranes. We showed that fusion peptides from influenza virus and HIV fusion machines do not induce fusion but can enhance PEG-mediated fusion of fusogenic membranes. We found that membrane curvature and PEG-associated osmotic stress both had dramatic effects on the kinetics of PEG-mediate fusion. We also developed new assays for lipid movement between membranes and for """"""""hydrophobic mismatch"""""""" associated with fusion intermediates. We have recently developed a simple model of the thermodynamics of a proposed reaction profile for the fusion process that suggests that molecular mismatch within the hydrophobic interior of intermediate structures in the fusion pathway may dominate the kinetics of the process. In the next funding period, we will test whether a simple kinetic scheme based on this model can account for the time courses of lipid and trapped aqueous compartment rearrangements during fusion. Next, we plan to use this model as a framework within which to address a unifying hypothesis for how the """"""""fusion peptide"""""""" and trans- membrane domains (TND) of fusion proteins contribute to biomembrane fusion. Finally, we will, in conjunction with the structural cell biology lab of Axel Brunger, develop a model system to define how SNARES and associated neuronal or intracellular fusion proteins influence the mechanism of PEG- mediate vesicle fusion. The results will shed light on the mechanisms of such natural cell fusion processes as endocytosis, exocytotic excretion, protein sorting, and viral budding and infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032707-18
Application #
6623887
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Chin, Jean
Project Start
1983-12-01
Project End
2006-03-31
Budget Start
2003-04-01
Budget End
2004-03-31
Support Year
18
Fiscal Year
2003
Total Cost
$271,600
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Biochemistry
Type
Schools of Medicine
DUNS #
608195277
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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