Lipid-Coated Gold Nanoparticles and FRET Allow Sensitive Monitoring of Liposome Clustering Mediated by the Synaptotagmin-7 C2A Domain.

Synaptotagmin (Syt) family proteins contain tandem C2 domains, C2A and C2B, which insert into anionic membranes in response to increased cytosolic Ca2+ concentration and facilitate exocytosis in neuronal and endocrine cells. The C2A domain from Syt7 binds lipid membranes much more tightly than the corresponding domain from Syt1, but the implications of this difference for protein function are not yet clear. In particular, the ability of the isolated Syt7 C2A domain to initiate membrane apposition and/or aggregation has been previously unexplored. Here, we demonstrate that Syt7 C2A induces apposition and aggregation of liposomes using Förster resonance energy transfer (FRET) assays, dynamic light scattering, and spectroscopic techniques involving lipid-coated gold nanoparticles (LCAuNPs). Protein-membrane binding, membrane apposition, and macroscopic aggregation are three separate phenomena with distinct Ca2+ requirements: the threshold Ca2+ concentration for membrane binding is lowest, followed by apposition and aggregation. However, aggregation is highly sensitive to protein concentration and can occur even at submicromolar Syt7 C2A; thus, highly sensitive assays are needed for measuring apposition without complications arising from aggregation. Notably, the localized surface plasmon resonance of the LCAuNP is sensitive to ≤10 nM Syt7 C2A concentrations. Furthermore, when the LCAuNPs were added into a FRET-based liposome apposition assay, the resultant energy transfer increased; possible explanations are discussed. Overall, LCAuNP-based methods allow for highly sensitive detection of protein-induced membrane apposition under conditions that miminize large-scale aggregation.

Lateral diffusion of proteins on supported lipid bilayers: additive friction of synaptotagmin 7 C2A-C2B tandem domains.

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.

Hydrophobic contributions to the membrane docking of synaptotagmin 7 C2A domain: mechanistic contrast between isoforms 1 and 7.

Synaptotagmin (Syt) triggers Ca(2+)-dependent membrane fusion via its tandem C2 domains, C2A and C2B. The 17 known human isoforms are active in different secretory cell types, including neurons (Syt1 and others) and pancreatic ß cells (Syt7 and others). Here, quantitative fluorescence measurements reveal notable differences in the membrane docking mechanisms of Syt1 C2A and Syt7 C2A to vesicles comprised of physiological lipid mixtures. In agreement with previous studies, the Ca(2+) sensitivity of membrane binding is much higher for Syt7 C2A. We report here for the first time that this increased sensitivity is due to the slower target membrane dissociation of Syt7 C2A. Association and dissociation rate constants for Syt7 C2A are found to be ~2-fold and ~60-fold slower than Syt1 C2A, respectively. Furthermore, the membrane dissociation of Syt7 C2A but not Syt1 C2A is slowed by Na(2)SO(4) and trehalose, solutes that enhance the hydrophobic effect. Overall, the simplest model consistent with these findings proposes that Syt7 C2A first docks electrostatically to the target membrane surface and then inserts into the bilayer via a slow hydrophobic mechanism. In contrast, the membrane docking of Syt1 C2A is known to be predominantly electrostatic. Thus, these two highly homologous domains exhibit distinct mechanisms of membrane binding correlated with their known differences in function.