title: Lateral diffusion of proteins on supported lipid bilayers: additive friction of synaptotagmin 7 C2A-C2B tandem domains.
abstract: The synaptotagmin (Syt) family of proteins contains tandem C2 domains, C2A and C2B, which bind membranes in the presence of Ca(2+) to trigger vesicle fusion during exocytosis. Despite recent progress, the role and extent of interdomain interactions between C2A and C2B in membrane binding remain unclear. To test whether the two domains interact on a planar lipid bilayer (i.e., experience thermodynamic interdomain contacts), diffusion of fluorescent-tagged C2A, C2B, and C2AB domains from human Syt7 was measured using total internal reflection fluorescence microscopy with single-particle tracking. The C2AB tandem exhibits a lateral diffusion constant approximately half the value of the isolated single domains and does not change when additional residues are engineered into the C2A-C2B linker. This is the expected result if C2A and C2B are separated when membrane-bound; theory predicts that C2AB diffusion would be faster if the two domains were close enough together to have interdomain contact. Stopped-flow measurements of membrane dissociation kinetics further support an absence of interdomain interactions, as dissociation kinetics of the C2AB tandem remain unchanged when rigid or flexible linker extensions are included. Together, the results suggest that the two C2 domains of Syt7 bind independently to planar membranes, in contrast to reported interdomain cooperativity in Syt1.
authors.affiliation: Department of Chemistry, University of Colorado Denver , Denver, Colorado 80217, United States.
authors.forename: Joseph K
authors.initials: JK
authors.lastname: Vasquez
authors.forename: Kan
authors.initials: K
authors.lastname: Chantranuvatana
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Giardina
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Coffman
authors.forename: Jefferson D
authors.initials: JD
authors.lastname: Knight
mesh_terms: D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D050863:Synaptotagmin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050857:Synaptotagmins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D008051:Lipid Bilayers; C494310:SYT1 protein, human; C494302:SYT7 protein, human; D050863:Synaptotagmin I; D050857:Synaptotagmins