Structure and Assembly Dynamics of FtsZ

FtsZ 的结构和装配动力学

• 批准号: 6605635
• 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/6605635
• 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（由申请人提供）：FtsZ是微管蛋白的同源物，是细菌细胞分裂的主要细胞骨架蛋白。FtsZ在细菌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地。最终，z形环收缩使细胞分裂。在体外，FtsZ组装成长而直的原丝（pfs），可以结合成pf对和薄片。我们最近提出了一种FtsZ pfs等轴组装的模型，这与肌动蛋白和微管的协同组装有很大的不同。该模型预测了pfs与GTP水解耦合的快速破碎和退火。利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正在快速翻转，中间时间为30秒。这与我们对等轴装配模型的期望是一致的。然而，该模型目前主要是基于pf长度的预测，必须获得证实或修改它的实验数据。最重要的是直接测量FtsZ亚基在pf中的相互作用亲和力。它是按照理论预测的nM量级，还是按照其他观察预测的uM量级？我们建议使用荧光各向异性来测量标记亚基与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，以及在不同FtsZ浓度下组装的pfs的长度。在一些研究中，我们将生产帽突变体，这些突变体在一端或另一端被阻止组装。它们结合成异源二聚体的反应应该比组装完整的pfs要简单得多。帽突变体也将用于体外研究GTPase的机制。作为这些体外研究的补充，我们将把FRAP在体内动态的研究扩展到新的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-它是单体还是pfs？我们将通过扩散测量来确定。总的来说，我们的目标是在体外完整地表征FtsZ原丝组装的生物物理特性，并通过FRAP对体内组装动力学和功能进行补充分析。