Membrane fusion is a critical and ubiquitous phenomenon in biology. However, the mechanism of biomembrane fusion remains one of the central mysteries of membrane biology. The """"""""stalk"""""""" theory is widely cited as the basis for biological membrane fusion, yet there is no direct evidence that even pure lipid membrane fusion occurs via this mechanism. There is evidence that both lipid bilayer fusion and bilayer/inverted phase transitions proceed via closely-related mechanisms, involving the same intermediate structures. Dr. Caffrey will use this correspondence to test two aspects of the stalk theory as a basis for biomembrane fusion, and also test a hypothesis about the role of transmembrane domains of fusion-inducing proteins. First, he will use a unique experimental system to attempt direct determination of fusion intermediate structure, using time-resolved x-ray diffraction. Recent time-resolved cryoelectron microscopy results show that transient, ordered arrays of fusion intermediates exist under special circumstances in systems undergoing the bilayer/inverted hexagonal phase transition. In this study Dr. Caffrey will establish conditions that increase the size of and order in these arrays, and will attempt to determin directly the intermediate structure using time-resolved x-ray diffraction. This would be the first direct demonistration of a fusion mechanism. Second, the stalk theory predicts that the membrane fusion and the rate of the bilayer/inverted cubic phase transition in bulk lipid/water systems are controlled by the rate of decay of the same intermediate structure. If the stalk theory is correct, reducing the bilayer rupture tension should increase both rates in parallel. Transmembrabe peptides have been shown to substantially reduce this rupture tension at low concentrations in lipid membranes. Therefore, Dr. Caffrey and collegues will add traces of carefully characterized transbilayer peptides to lipid systems, measure their effect on the membrane tensions, and measure the rates of both the phase transition and the rate of membrane fusion in unilmaellar liposomes containing these peptides. The latter experiment will also test the hypothesis that the transmembrane domains of fusion-catalyzing proteins in viruses are imoportant to fusion activity because they serve to destabilize the same sort of fusion itermediate. In influenza virus hemagglutinin (HA), the transmembrane domain is crtical for fusion activity: substituting a lipid anchor for this domaim destroys HA fusion activity, but the lipid mixing activity inact. Such activity is consistent with Dr. Caffrey's hypothesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM056969-01
Application #
2464854
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1998-01-01
Project End
2001-12-31
Budget Start
1998-01-01
Budget End
1998-12-31
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Ohio State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
098987217
City
Columbus
State
OH
Country
United States
Zip Code
43210
Misquitta, Lisa V; Misquitta, Yohann; Cherezov, Vadim et al. (2004) Membrane protein crystallization in lipidic mesophases with tailored bilayers. Structure 12:2113-24
Cherezov, V; Siegel, D P; Shaw, W et al. (2003) The kinetics of non-lamellar phase formation in DOPE-Me: relevance to biomembrane fusion. J Membr Biol 195:165-82
Ravelli, Raimond B G; Leiros, Hanna-Kirsti Schroder; Pan, Baocheng et al. (2003) Specific radiation damage can be used to solve macromolecular crystal structures. Structure 11:217-24
Caffrey, Martin (2003) Membrane protein crystallization. J Struct Biol 142:108-32
Koynova, Rumiana; Caffrey, Martin (2002) An index of lipid phase diagrams. Chem Phys Lipids 115:107-219
Cherezov, Vadim; Riedl, Ken M; Caffrey, Martin (2002) Too hot to handle? Synchrotron X-ray damage of lipid membranes and mesophases. J Synchrotron Radiat 9:333-41
Cherezov, Vadim; Qiu, Hong; Pector, Veronique et al. (2002) Biophysical and transfection studies of the diC(14)-amidine/DNA complex. Biophys J 82:3105-17
Brzustowicz, Michael R; Cherezov, Vadim; Caffrey, Martin et al. (2002) Molecular organization of cholesterol in polyunsaturated membranes: microdomain formation. Biophys J 82:285-98
Brzustowicz, Michael R; Cherezov, Vadim; Zerouga, Mustapha et al. (2002) Controlling membrane cholesterol content. A role for polyunsaturated (docosahexaenoate) phospholipids. Biochemistry 41:12509-19
Cherezov, Vadim; Clogston, Jeffrey; Misquitta, Yohann et al. (2002) Membrane protein crystallization in meso: lipid type-tailoring of the cubic phase. Biophys J 83:3393-407

Showing the most recent 10 out of 23 publications