Characterization of constitutively active Arp2/3

组成型活性 Arp2/3 的表征

• 批准号: 6937329
• 负责人: Paul Dalhaimer
• 金额: $ 4.4万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6937329
• 关键词: Schizosaccharomyces pombe; actins; cell motility; cell nucleus; chemical kinetics; conformation; crosslink; cysteine; fluorescence microscopy; intermolecular interaction; microfilaments; molecular dynamics; nucleotides; postdoctoral investigator; posttranslational modifications; protein structure; spectrometry; thermodynamics

My goal is a quantitative understanding of the activation of Arp2/3 complex, a protein assembly instrumental in cellular motility. I propose three lines of experiments to understand how activators change the conformation of Arp2/3 complex and how this conformational change influences actin filament nucleation and branching in vivo and in vitro. . Use disulfide crosslinking between cysteine residues engineered into fission yeast Arp2/3 complex to create constitutively active and inactive Arp2/3 complex. I will compare native Arp2/3 complex and these two frozen conformations in spectroscopic and single particle fluorescence microscopy assays to analyze the kinetics and thermodynamics of Arp2/3 complex activation by nucleation promoting factors such as the VGA regions of WASp and Scar, actin monomers, and pre-existing actin filaments. . Use high-resolution 3D fluorescence microscopy to characterize the effects of a panel of mutations on the ability of Arp2/3 complex to support actin filament nucleation in live fission yeast. Use simulations to study Arp2/3 subunit mechanisms involved in nucleotide binding and activation.

我的目标是对ARP2/3复合物的激活的定量理解，ARP2/3复合物是一种在细胞运动中造成的蛋白质组件。我提出了三条实验线，以了解激活剂如何改变ARP2/3复合物的构象，以及这种构象变化如何影响肌动蛋白丝核和体内和体外分支。 。在裂变酵母ARP2/3复合物中的半胱氨酸残基之间使用二硫键交联，以创建组成性活跃和不活跃的ARP2/3复合物。我将比较天然ARP2/3复合物以及光谱和单个颗粒荧光显微镜测定中的这两个冷冻构象，以分析通过促进因子促进因子，例如WASP和Scarp和Scarp，Actin Monomers，Pre-Pre-Monomers和Pre-Pre-excatinsisting actinements filements contements of ARP2/3复合物激活的动力学和热力学。 。使用高分辨率3D荧光显微镜表征突变对 ARP2/3复合物支持活裂变酵母中肌动蛋白丝成核的能力。 使用模拟研究参与核苷酸结合和激活的ARP2/3亚基机制。