Probing the Structure and Contraction Mechanism of the E. coli FtsZ-ring.

描述（由申请人提供）：本申请的长期目标是阐明细菌细胞分裂的精确分子控制机制。这里描述的以FtsZ蛋白为中心的项目是实现这一目标的第一步。FtsZ在细菌物种中高度保守，已被确定为抗菌治疗的新型药物靶标。在体内，FtsZ在内陷膜的前缘组装成超分子环状结构（因此称为Z环）以驱动细胞分裂。该项目的目标是确定Z环的结构，特别是Z环内原丝的排列（目标1），并确定Z环的收缩机制（目标2）。在目标1中，使用基于单分子的超分辨率成像技术，以30 nm的空间分辨率对Z环进行成像。提出了一种FtsZ原丝的三维束状排列而不是二维扁平带状排列。这种安排模式将进一步验证检查三个预测源于该模型。这一目标的最终产物是一个高分辨率的Z环结构模型，这将有助于理解Z环的收缩机制。在目标2中，将研究两种竞争的Z环收缩机制-分解与缩合。将使用超分辨率成像技术测量收缩期间Z形环的收缩速度。然后将该速度与基于Z形环的分解速率和密度变化的单分子测量的计算速度进行比较。GTP水解在Z环收缩中的作用也将使用FtsZ GTP酶突变体进行检查。这些实验，结合将在目标1中建立的结构模型，将为Z环收缩的分子机制提供重要的见解。 所提出的工作的成功完成将提供：（1）高分辨率Z环结构模型;（2）该结构如何收缩的机制和（3）一组创新的体内单分子测定。 公共卫生相关性：本研究的目的是了解细菌细胞如何使用中央超分子组装（Z环）进行分裂。由于Z环对于细菌王国的生存和保存至关重要，因此更好的理解将促进新抗生素的更有效开发，以对抗病原菌引起的传染病。

DESCRIPTION (provided by applicant): The long-term goal of this application is to unravel mechanisms underlying the precise molecular controls of bacterial cell division. The project described here, centered on the FtsZ protein, is the first step toward this goal. FtsZ is highly conserved across bacterial species and has been identified as a novel drug target for antimicrobial therapy. In vivo FtsZ assembles into a supramolecular ring-like structure (hence named the Z-ring) at the leading edge of the invaginating membrane to drive cell division. The objective of this project is to determine the structure of the Z-ring, in particular the arrangement of protofilaments inside the Z-ring (Aim 1), and to identify the contraction mechanism of the Z-ring (Aim 2). In Aim 1, using a single-molecule based superresolution imaging technique the Z-ring was imaged with a 30-nm spatial resolution. A 3D bundle arrangement of FtsZ protofilaments instead of a 2D flat ribbon was proposed. Such an arrangement model will be further verified by examining three predictions stemmed from the model. The final product of this aim is a high resolution Z-ring structural model that will be instrumental in understanding the contraction mechanism of the Z-ring. In Aim 2, two competing Z-ring contraction mechanisms-disassembly vs. condensation-will be examined. The contraction speed of the Z-ring during the constriction period will be measured using the superresolution imaging technique. This speed will then be compared with the calculated speed based on single-molecule measurements of the disassembly rate and density change of the Z-ring. The role of GTP hydrolysis in Z-ring contraction will also be examined using an FtsZ GTPase mutant. These experiments, combined with the structural model that will be established in Aim 1, will provide important insight into the molecular mechanism of Z-ring contraction. Successful completion of the proposed work will provide: (1) a high-resolution Z- ring structural model; (2) a mechanism of how this structure contracts and (3) a set of innovative in vivo single-molecule assays. PUBLIC HEALTH RELEVANCE: The goal of this study is to understand how bacterial cells divide using a central supramolecular assembly, the Z-ring. As the Z-ring is essential for survival and conserved across the bacterial kingdom, a better understanding will promote more effective development of new antibiotics to combat infectious diseases caused by pathogenic bacteria.