Structure and Assembly Dynamics of FtsZ

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在细菌的中心形成一个环，在细胞周期的大部分时间里保持在适当的位置。最终，Z环收缩以分裂细胞。在体外，FtsZ组装成长的、直的原丝(PF)，可以结合成PF对和片状。我们最近提出了一种FtsZ PFS等线组装的模型，这与肌动蛋白和微管的协同组装有很大不同。该模型预测了PFS在GTP水解过程中的快速碎裂和退火过程。利用荧光漂白后恢复(FRAP)进行体内分析，我们还确定了Z环中的FtsZ正在迅速翻转，半衰期为30秒。这与我们从等键组装模型中的预期一致。然而，目前的模型主要是基于对PF长度的预测，并且必须获得实验数据来证实或修正它。最重要的是直接测量PF中FtsZ亚基的相互作用亲和力。它是理论所预言的纳米量级，还是其他观测所预言的纳米量级？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Biaccore进行更多的定量分析，以确定PF组装的Kd，并有望确定在不同FtsZ浓度下组装的PFS的长度。对于几项研究，我们将产生在一端或另一端被阻止组装的帽子突变体。它们结合成杂二聚体的反应应该比组装完整的PFS简单得多。这些帽子突变体也将被用于体外研究GTP酶的机制。作为对这些体外研究的补充，我们将把我们的体内动力学的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。总之，我们的目标是完整地描述FtsZ原丝在体外组装的生物物理学，并通过FRAP对体内组装动力学和功能进行补充分析。

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.