The function and organization of MreB filaments

描述（由申请人提供）：与真核细胞一样，细菌具有多种细胞骨架元素。这些细菌细胞骨架元件之一，MreB，真核肌动蛋白的同源物，已被证明参与维持细胞形状和蛋白质定位。MreB控制细胞壁合成以维持细胞形状或直接蛋白定位的机制尚不清楚。MreB在结构上与肌动蛋白相似，两种蛋白在细胞内形成细丝。然而，MreB在纯化时并不像在体内那样形成螺旋结构。已知真核肌动蛋白有数百种相互作用蛋白，但MreB只确定了一种相互作用蛋白（RodZ），这表明可能存在未知的辅助蛋白。为了更好地理解MreB的功能，我建议将重点放在识别和描述其交互伙伴上。特别是，我在大肠杆菌中开发了一种双分子荧光互补（BiFC）试验，这将使研究mreb蛋白在体内的相互作用成为可能。我将首先使用该试验对MreB细丝进行功能分析，尽管MreB点突变体的表达与自身结合增强或减少，然后进行体外聚合试验和电子显微镜检查以确认这些结果。RodZ是一种已知的结合伙伴，它在纤维稳定中的作用也将被研究。其次，我将探讨MreB对细胞正常生长的作用。我将测试MreB作为细胞壁合成复合物支架的假设。我还将探索MreB与细胞代谢蛋白相互作用以正确定位它们的假设。将对MreB表面暴露的残基进行丙氨酸扫描诱变，以确定MreB上是否存在与其他蛋白质结合的单一相互作用结构域。该提案的结果将创造关于MreB功能的新知识，然后可以应用于该领域的新型抗菌化合物的创建。

DESCRIPTION (provided by applicant): Like eukaryotic cells, bacteria have multiple cytoskeletal elements. One of these bacterial cytoskeleton elements, MreB, a homolog of eukaryotic actin, has been shown to be involved in maintaining cell shape and protein localization. The mechanism by which MreB controls cell wall synthesis to maintain cell shape or direct protein localization remains unclear. MreB resembles actin structurally, and both proteins form filaments within cells. However, MreB does not form a helical structure when purified as it does in vivo. Hundreds of interacting proteins are known for eukaryotic actin, but only one interacting partner (RodZ) has been confirmed for MreB, indicating there are probably unknown accessory proteins. In order to better understand MreB function, I propose to focus on identifying and characterizing its interacting partners. In particular, I have developed a bimolecular fluorescent complementation (BiFC) assay in Escherichia coli, which will enable the study of MreB-protein interactions in vivo. I will initially use the assay to perform a functional analysis of MreB filaments, though the expression of MreB point mutants with enhanced or reduced binding to itself, followed by in vitro polymerization assays and electron microscopy to confirm these results. The role of RodZ, a known binding partner, will also be looked at for its role in filament stabilization. Secondly, I will look at the role of MreB for proper cell growth. Iwill test the hypothesis that MreB acts as a scaffold for a cell wall synthesis complex. I will also being to explore the hypothesis that MreB interacts with cellar metabolic proteins to properly localize them. An alanine scanning mutagenesis will be performed on surface exposed residues of MreB to determine if there is a single interaction domain on MreB that other proteins bind. The results of this proposal will create new knowledge of the functionality of MreB, which can then be applied by the field to the creation of novel antimicrobial compounds.