Frictional Additivity of Lateral Diffusion on Supported Bilayers: Influence of Linker Length in 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 Ca2+. The precise mechanism by which Syt participates in membrane fusion events is not completely understood, and the role of interactions between C2A and C2B in membrane binding and fusion is unclear. To test whether the two domains interact with each other on a planar lipid bilayer, diffusion constants of fluorescent-tagged C2A, C2B, and tandem C2AB domains from Syt7 were determined using total internal reflection fluorescence microscopy with single-particle tracking. As expected, the tandem domain diffuses significantly more slowly than the single domains. However, the diffusion constant of the C2AB tandem is significantly faster than predicted by simple summation of the individual domains' frictional coefficients. This result suggests that either (a) the short interdomain linker holds the two domains in close proximity to each other in the plane of the bilayer, at a separation distance shorter than the free draining limit, and/or (b) the two domains directly interact with each other when membrane-bound. In order to distinguish between these two possibilities, several mutant versions of the tandem domain with extended C2A-C2B linker regions were purified. Preliminary results indicate these mutants continue to diffuse similarly to the native C2AB tandem and faster than predicted based on frictional additivity, even at linker distances expected to exceed the free draining limit. These results suggest that C2A and C2B interact with each other when bound to a membrane in a manner that is independent of C2A−C2B linker length. Current experiments in this ongoing project probe the nature of the C2A-C2B interaction and may provide insight into the membrane fusion activity of this protein.