Structure and Assembly Dynamics of FtsZ

FtsZ 的结构和装配动力学

• 批准号: 6521821
• 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/6521821
• 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，在大多数细胞周期中，该环保持在适当的位置。最终，Z形会收缩以分裂细胞。在体外，FTSZ组装成长长的直丝（PFS），可以将其关联成PF对和床单。我们最近提出了一个模型，其中FTSZ PFS组装iSodesmammammammammal，这与肌动蛋白和微管的合作组装大不相同。该模型预测了与GTP水解耦合的PFS的快速分裂和退火。使用光漂白后的荧光回收（FRAP）进行体内分析，我们还确定Z环中的FTSZ在中场时间为30秒，迅速地翻转过来。这与我们对iSodesmic组装模型的期望是一致的。但是，当前该模型主要基于PF长度的预测，并且必须获得确认或修改它的实验数据。最重要的是直接衡量PF中FTSZ亚基的相互作用亲和力。是按照理论所预测的NM的顺序，还是其他观察结果所预测的UM？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进行更定量的分析，以确定PF组装的KD，并希望以不同的FTSZ浓度组装的PF长度。对于几项研究，我们将产生盖帽突变体，这些突变体在一个或另一端被阻止用于组装。与完整PFS组装相比，它们与异二聚体的关联应该是一个简单得多的反应。盖突变体还将用于在体外研究GTPase机制。与这些体外研究相辅相成，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细菌细胞质中FTSZ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，我们的目的是在体外的FTSZ原丝组装的生物物理学以及体内组装动力学和功能的互补分析中进行完整表征。