Structure and Assembly Dynamics of FtsZ

FtsZ 的结构和装配动力学

• 批准号: 6914857
• 负责人: HAROLD P ERICKSON
• 金额: $ 38.5万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6914857
• 关键词: bacterial genetics; bacterial proteins; bioassay; bioenergetics; fluorescence polarization; green fluorescent proteins; guanosinetriphosphatases; membrane proteins; microorganism growth; molecular assembly /self assembly; molecular size; plasmids; protein structure function; restriction mapping; site directed mutagenesis; temperature sensitive mutant; tubulin

DESCRIPTION (provided by applicant): FtsZ, a homolog of tubulin, is the major cytoskeletal protein of bacterial cell division. FtsZ forms a ring around the center of the bacterium, which remains in place for most of the cell cycle. Ultimately the Z-ring constricts to divide the cell. In vitro, FtsZ assembles into long, straight protofilaments (pfs) that can associate into pf pairs and sheets. We have recently proposed a model in which FtsZ pfs assemble isodesmically, which is very different from the cooperative assembly of actin and microtubules. This model predicts a rapid fragmentation and annealing of pfs coupled to GTP hydrolysis. Using fluorescence recovery after photobleaching (FRAP) for in vivo analysis, we have also determined that FtsZ in the Z-ring is turning over rapidly, with a 30 sec halftime. This is consistent with our expectations from the isodesmic assembly model. However, that model at present is based largely on predictions from pf length, and it is essential to obtain experimental data confirming or modifying it. Most important is a direct measure of the interaction affinity of FtsZ subunits in the pf. Is it on the order of nM as predicted by the theory, or on the order of uM as predicted by other observations? We propose to use fluorescence anisotropy to measure the association of labeled subunits to pfs and estimate the Kd. We will then use the Biacore for more quantitative analysis, to determine the Kd for pf assembly, and hopefully the length of pfs assembled at different FtsZ concentrations. For several studies we will produce cap mutants that are blocked for assembly at one or the other ends. Their association into heterodimers should be a much simpler reaction than assembly of full pfs. The cap mutants will also be used to study the GTPase mechanism in vitro. Complementing these in vitro studies, we will extend our FRAP study of in vivo dynamics to new FtsZ mutants and accessory proteins. An important question that we can now address by FRAP is the state of assembly of FtsZ in the bacterial cytoplasm - is it monomers or pfs? We will determine this by diffusion measurements. Overall, we are aiming for a complete characterization of the biophysics of FtsZ protofilament assembly in vitro, and complementary analysis by FRAP of assembly dynamics and function in vivo.

描述（申请人提供）：FtsZ是微管蛋白的同系物，是细菌细胞分裂的主要细胞骨架蛋白。FtsZ在细菌的中心周围形成一个环，在细胞周期的大部分时间里都保持在原位。最终，Z环收缩以分裂细胞。在体外，FtsZ组装成长而直的原丝（pfs），可以结合成pf对和片。我们最近提出了一个模型，其中FtsZ pfs组装isodesmically，这是非常不同的肌动蛋白和微管的合作组装。该模型预测了一个快速的碎片和退火的pfs耦合到GTP水解。使用光漂白后荧光恢复（FRAP）进行体内分析，我们还确定了Z环中的FtsZ快速翻转，半衰期为30秒。这与我们对等键组装模型的期望是一致的。然而，该模型目前主要是基于预测从pf的长度，它是必要的，以获得实验数据证实或修改it. Most重要的是一个直接测量的相互作用亲和力的FtsZ亚基的pf。它是在nM的顺序预测的理论，或在uM的顺序预测的其他观测？我们建议使用荧光各向异性来测量标记的亚基与pfs的关联并估计Kd。然后，我们将使用Biacore进行更多的定量分析，以确定pf组装的Kd，并希望在不同的FtsZ浓度下组装的pfs的长度。在几项研究中，我们将产生在一端或另一端被阻止组装的cap突变体。它们结合成异二聚体的反应应该比组装完整的pfs简单得多。cap突变体也将用于体外研究GT3机制。补充这些体外研究，我们将扩展我们的FRAP研究在体内的动态新的FtsZ突变体和辅助蛋白。我们现在可以通过FRAP解决的一个重要问题是FtsZ在细菌细胞质中的组装状态-是单体还是pfs？我们将通过扩散测量来确定这一点。总的来说，我们的目标是一个完整的生物物理特性的FtsZ原丝组装在体外，并补充分析FRAP的组装动力学和功能在体内。