The synaptotagmin (Syt) family of proteins contains tandem C2 domains, C2A and C2B, which bind membranes in the presence of Ca2+ 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.

No abstract

dynamic structural model for the TCR ab TM complex and how it associates with the CD3 zz subunit. Additionally, the interaction of the TCR with its lipid environment may also regulate the organization of the TCR-CD3 TM complex. We will also discuss simulation of the TCR TM region in complex lipid bilayers that resemble plasma membranes. Our results suggest preferential interactions of the TCR TM region with cholesterol and anionic lipids in the bilayer.

membrane and the atomic structure has been resolved by X-ray crystallography. The hydrophobic thickness of the protein observed in the structure 29.7A is considerably thinner than that predicted for the membrane 35.5A suggesting a hydrophobic mismatch. This hydrophobic mismatch is expected to aid protein-protein interactions and result in lipid sorting. Furthermore the surface of the protein has several cationic groups that could result in specific interactions with anionic lipid species. In order to investigate the local environment of Aquaporin Z in native E. coli membranes, and so understand the degree of lipid sorting and local protein-protein interactions, we have isolated Styrene-Maleic Acid Lipid Particles. These particles are believed to contain small portions of membrane isolated by the detergent action of the polymer. We have analysed particles isolated from E.coli membranes and containing Aquaporing Z for size, lipid and protein content. We have compared these experimental results to results obtained from coarse grained molecular dynamic simulations. These results are interpreted in terms of the interactions between the aquaporin and its environment.