| Popis: |
Synaptotagmin 1 (Syt1), a Ca2+ sensor involved in exocytosis in pre-synaptic neurons, contains a characteristic tandem C2 sequence (C2AB) which inserts into anionic membranes in the presence of Ca2+. Previous studies have shown that the C2AB fragment of Syt1 inserts into target membranes more deeply than either individual C2A or C2B domain, suggesting cooperative interaction between C2A and C2B in the membrane-docked state. This behavior stands in apparent contrast to that of another family member, Syt7, whose C2A and C2B domains have been shown to bind membranes independently. To compare the cooperative behaviors of the C2 domains in these two isoforms, the dissociation kinetics (off-rates) of protein-liposome complexes were measured upon addition of the Ca2+ chelator EDTA using stopped-flow fluorescence spectroscopy. Using liposomes composed of a 1:1 mixture of phosphatidylcholine and phosphatidylserine (PC/PS), the Syt1 C2AB tandem domain exhibits a much slower off-rate than either of the single domains. In contrast, dissociation kinetics for Syt7 C2AB were best fit to a two-step model in which each rate constant matches those from the individual domains. This preliminary result supports the presence of interdomain interactions in the membrane-docked state of Syt1 C2AB but not Syt7 C2AB. These experiments were performed with protein domains purified using affinity chromatography with high-salt washes and verified to be >95% free of nucleic acid contaminants; ongoing work aims to assess effects of polyanions on this apparent cooperative behavior, including ion-exchange steps in the protein purifications. |
