Dataset Information

Conformation and membrane position of the region linking the two C2 domains in synaptotagmin 1 by site-directed spin labeling.

ABSTRACT: Synaptotagmin 1 (syt1) is an integral membrane protein localized on the synaptic vesicle that acts as the Ca(2+) sensor for neuronal exocytosis. Synaptotagmin 1 contains two C2 domains, C2A and C2B, which bind Ca(2+) ions, membranes, and SNAREs. Here, site-directed spin labeling (SDSL) was used to determine the position and dynamics of the region that links the two C2 domains in a water soluble construct encompassing the two C2 domains (syt1C2AB). An analysis of the EPR line shapes from this region indicates that the linker is flexible and unstructured when syt1 is in solution or bound to lipid bilayers. The nanosecond dynamics of the linker does not change, in the presence or absence of Ca(2+), suggesting that there is no Ca(2+)-dependent intramolecular association between the two domains. When syt1C2AB is membrane-bound, the position of the linker relative to the membrane interface was determined by measuring parameters for the collision of the spin-labeled syt1C2AB mutants with both soluble and membrane-bound Ni(II) chelates. These data indicate that the linker does not penetrate the membrane surface but lies approximately 7-10 A from the bilayer surface. In addition, the linker remains flexible when syt1C2AB binds to the SNARE complex, indicating that direct interactions between this linker and the SNAREs do not mediate association. These data suggest that the two C2 domains of syt1 interact independently on the membrane interface, or when bound to SNAREs.

SUBMITTER: Huang H

PROVIDER: S-EPMC2661311 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Conformation and membrane position of the region linking the two C2 domains in synaptotagmin 1 by site-directed spin labeling.

Huang Hao H Cafiso David S DS

Biochemistry 20081101 47

Synaptotagmin 1 (syt1) is an integral membrane protein localized on the synaptic vesicle that acts as the Ca(2+) sensor for neuronal exocytosis. Synaptotagmin 1 contains two C2 domains, C2A and C2B, which bind Ca(2+) ions, membranes, and SNAREs. Here, site-directed spin labeling (SDSL) was used to determine the position and dynamics of the region that links the two C2 domains in a water soluble construct encompassing the two C2 domains (syt1C2AB). An analysis of the EPR line shapes from this reg ...[more]

PMID: 18956883

Similar Datasets

Project description:The C2 domain is a conserved signaling motif that triggers membrane docking in a Ca(2+)-dependent manner, but the membrane docking surfaces of many C2 domains have not yet been identified. Two extreme models can be proposed for the docking of the protein kinase C alpha (PKC alpha) C2 domain to membranes. In the parallel model, the membrane-docking surface includes the Ca(2+) binding loops and an anion binding site on beta-strands 3-4, such that the beta-strands are oriented parallel to the membrane. In the perpendicular model, the docking surface is localized to the Ca(2+) binding loops and the beta-strands are oriented perpendicular to the membrane surface. The present study utilizes site-directed fluorescence and spin-labeling to map out the membrane docking surface of the PKC alpha C2 domain. Single cysteine residues were engineered into 18 locations scattered over all regions of the protein surface, and were used as attachment sites for spectroscopic probes. The environmentally sensitive fluorescein probe identified positions where Ca(2+) activation or membrane docking trigger measurable fluorescence changes. Ca(2+) binding was found to initiate a global conformational change, while membrane docking triggered the largest fluorescein environmental changes at labeling positions on the three Ca(2+) binding loops (CBL), thereby localizing these loops to the membrane docking surface. Complementary EPR power saturation measurements were carried out using a nitroxide spin probe to determine a membrane depth parameter, Phi, for each spin-labeled mutant. Positive membrane depth parameters indicative of membrane insertion were found for three positions, all located on the Ca(2+) binding loops: N189 on CBL 1, and both R249 and R252 on CBL 3. In addition, EPR power saturation revealed that five positions near the anion binding site are partially protected from collisions with an aqueous paramagnetic probe, indicating that the anion binding site lies at or near the surface of the headgroup layer. Together, the fluorescence and EPR results indicate that the Ca(2+) first and third Ca(2+) binding loops insert directly into the lipid headgroup region of the membrane, and that the anion binding site on beta-strands 3-4 lies near the headgroups. The data support a model in which the beta-strands are tilted toward the parallel orientation relative to the membrane surface.

Dataset Information

Conformation and membrane position of the region linking the two C2 domains in synaptotagmin 1 by site-directed spin labeling.

Publications

Conformation and membrane position of the region linking the two C2 domains in synaptotagmin 1 by site-directed spin labeling.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets