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是微管蛋白的同系物，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位。最终，Z 形环收缩以分裂细胞。在体外，FtsZ 组装成长而直的原丝 (pfs)，可以结合成 pf 对和片状。我们最近提出了一个 FtsZ pfs 等序列组装的模型，这与肌动蛋白和微管的协同组装有很大不同。该模型预测 pfs 的快速裂解和退火与 GTP 水解相结合。使用光漂白后荧光恢复 (FRAP) 进行体内分析，我们还确定 Z 环中的 FtsZ 正在快速翻转，中场时间为 30 秒。这与我们对等脉组装模型的期望一致。然而，目前该模型主要基于 pf 长度的预测，因此必须获得实验数据来证实或修改它。最重要的是直接测量 pf 中 FtsZ 亚基的相互作用亲和力。是理论预测的 nM 量级，还是其他观测预测的 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 进行更多定量分析，以确定 pf 组装的 Kd，并希望确定在不同 FtsZ 浓度下组装的 pfs 的长度。对于几项研究，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。它们与异二聚体的结合应该是比完整 pfs 的组装简单得多的反应。 cap 突变体还将用于体外研究 GTPase 机制。作为这些体外研究的补充，我们将把体内动力学的 FRAP 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 - 是单体还是 pfs？我们将通过扩散测量来确定这一点。总体而言，我们的目标是对 FtsZ 原丝体外组装的生物物理学进行完整表征，并通过 FRAP 对体内组装动力学和功能进行补充分析。