Reconstitution of membrane fusion sites. A total internal reflection fluorescence microscopy study of influenza hemagglutinin-mediated membrane fusion.

摘要: Abstract Influenza hemagglutinin (HA, strain A/PR/8/34) was purified and reconstituted into supported planar membranes in a two-step process: 1) HA by C12E8 detergent solubilization followed removal with Biobeads; (2) the then incorporated "viroplanes," i.e. which contained viral membrane proteins. This step accomplished spontaneous reaction of HA-proteoliposomes phospholipid monolayer that on quartz microscope slide. The reconstitution total internal reflection fluorescence microscopy (TIRFM) using fluorescein-labeled HA. By changing solution concentration HA, surface concentrations between 2.4 x 10(4) 4.3 monomers/micron 2 were reached. Greater than 90% all molecules oriented their ectodomain facing away from substrate toward large aqueous compartment measuring cell. Binding experiments conformation-sensitive monoclonal antibodies against established could undergo low pH-induced conformational change bilayer. vesicles containing fluorescent lipid analog N-(7-nitro-2,1,3-benzoxadiazol-4-yl)egg phosphatidylethanolamine also measured TIRFM. Vesicle binding promoted when sialic acid-containing gangliosides or negatively charged lipids included these target membranes. Membrane fusion bound monitored long range (over several micrometers) lateral diffusion coefficients layer recovery after photobleaching. did not fuse at pH 7.4, but efficient vesicle occurred viroplanes acidification environment 5 buffer. only observed exceeded critical threshold concentration. successful sites system opens new possibilities for studying intermediates localized spectroscopy microscopy.