MAPPING THE STRUCTURE OF SNARE

绘制圈套的结构

• 批准号: 8364003
• 负责人: PETER P BORBAT
• 金额: $ 0.8万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8364003
• 关键词: Cell membrane; Complex; Cysteine; Equilibrium; Eukaryota; Funding; Golgi Apparatus; Grant; Intracellular Membranes; Label; Maps; Measures; Membrane; Membrane Fusion; National Center for Research Resources; Principal Investigator; Process; Proteins; Research; Research Infrastructure; Resources; SNAP receptor; Source; Spectrum Analysis; Structure; Synapses; Technology; Transmembrane Domain; United States National Institutes of Health; Vesicle; Yeasts; cost; genetic regulatory protein; interest; monomer; mutant; syntaxin-2; target SNARE proteins; trafficking; vesicular SNARE proteins

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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供 该子项目的主要支持。 并且子项目的首席研究员可能是由其他来源提供的， 包括其他 NIH 来源的子项目可能列出的总成本。 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 SNARE 复合物的形成是细胞内膜融合的核心，这是真核生物中囊泡运输的必要步骤。囊泡相关 (v-) SNARE 和靶膜 (t-) SNARE 之间的关联导致形成卷曲的线圈核心。在由额外的调节蛋白和底物协调的复杂融合过程中，参与的 SNARE 蛋白的跨膜结构域 (TMD) 可能会在该过程中寡聚化。专注于突触 (v-) SNARE 蛋白 VAMP2 的 α 螺旋 TMD，将准备一组双标记半胱氨酸突变体，通过偶极光谱测量各个硝基氧之间的距离，以表征该结构域（<10 ¿）。 ）将通过 CW-ESR 测量，而更长的距离将通过 PDS（DQC-ESR）获得，随着研究的进展，残基之间的距离。还可以研究 VAMP2 的 TMD 以及由 VAMP2 的 Syntaxin-2 结构域和可溶性 (t-) SNARE-DTMD 形成的螺旋束，之前的 ESR 分析表明，目标膜 (t-) Sso1p 的单体和寡聚物之间存在平衡。 ) SNARE 参与酵母中从高尔基体到质膜的运输寡聚化的程度（如果有的话）将由 Ku-band DEER 使用单标记的 VAMP2 研究。突变体。