This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The formation of SNARE complex is central for intracellular membrane fusion, which is a necessary step of vesicular transport in eukaryotes. Association between vesicle-associated (v-) SNARE and target membrane (t-) SNARE results in the coiled coil core that holds together two membranes in the complex process of fusion orchestrated by additional regulatory proteins and substrates. The transmembrane domain (TMD) of participating SNARE proteins may oligomerize in the process. A current interest is focused on alpha-helical TMD of synaptic (v-) SNARE protein, VAMP2. A set of doubly-labeled cysteine mutants will be prepared to measure distances between respective nitroxides by dipolar spectroscopy to characterize this domain. Short distances (<10 ?) will be measured by CW-ESR, whereas longer distances will be obtained by PDS (DQC-ESR). As the study is progressing distances between the residues on TMD of VAMP2 and helical bundle formed by Syntaxin-2 domains of VAMP2 and soluble (t-) SNARE-DTMD could also be studied. Previous ESR analysis showed that there is equilibrium between the monomers and oligomers of Sso1p, a target membrane (t-) SNARE involved in the trafficking from Golgi to plasma membrane in yeast. The extent of oligomerization (if any) will be studied by Ku-band DEER using singly-labeled VAMP2 mutants.
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