Self-assembly and function of bacterial microcompartments

细菌细胞所拥有的超微结构明显多于人们通常所认识到的。一 最引人注目的例子是细菌微隔室(BMC)，大(即100+纳米) 包裹货运酶的蛋白质类复合体，催化体内的短代谢途径 像衣壳一样的壳。骨髓基质细胞使新陈代谢与宿主不相容，这一功能优势 它们的普遍存在证明了这一点。20%-30%的细菌基因组具有BMC样蛋白。尽管 在这种盛行的情况下，只有少数几种BMC具有特征性。最耐人寻味的公开问题之一 围绕着BMCs的是一个成熟的功能复合体是如何从蛋白质-蛋白质相互作用中产生的。 具体地说，组装、货物排序和化学计量的机制，以及坚固性、形状和 成熟复合体的大小不能用目前已知的定性知识来解释 蛋白质的相互作用。我们的工作目标是使用机械化的生化方法来 了解被称为α-羧体(α-CB)的骨髓基质细胞在体内的自组装和功能。这个 α-CB促进了许多细菌的自养生长，是第一个被表征的骨髓基质细胞，因为它的 生物化学分析的稳健性和易用性。因此，这是一个很好的模型系统来回答这些问题 公开的问题。初步数据表明，一种名为CsoS2的蛋白质对α-CB的形成是必不可少的 并且可以是驱动自组装的交互网络的中枢。我们建议使用生化和 生物物理工具，以绘制这些相互作用的分子决定因素图并定量地 了解多价态如何控制组装。CsoS2也是一种天生无序的蛋白质 拥有大量重复的序列元件。初步数据表明，CsoS2的这些区域 在确定α-CB尺寸方面起着重要作用。已知先天无序的蛋白质参与 在真核生物中起组织作用，但在原核生物中基本上没有特征。因此，我们建议 了解紊乱对CsoS2功能的意义及其重复性的一系列实验 在决定组装过程的结果时涉及到要素。最后，它长期以来一直是 假设骨髓基质细胞的行为像细胞器，并拥有与细胞不同的化学环境 胞浆。这一假设得到了间接数据的支持，但从未被直接测量过 由于实验上的挑战，在生化方面。在这里，我们提出了一系列实验来实现这一点 通过确定α-CB是否由于以下原因自然具有氧化管腔来进行体外测量 它的蛋白质外壳的作用。我们还将确定管腔的化学成分达到什么程度 影响自组装过程。如果成功，这些实验将提供新的机械洞察力 了解骨髓细胞如何组装和功能，以及更广泛地说，细菌超微结构之间的相互作用 和细菌生理学。

Bacterial cells possess significantly more ultrastructural organization than is typically appreciated. One of the most striking examples of this are bacterial microcompartments (BMCs), large (i.e. 100+ nm) proteinaceous complexes that encapsulate cargo enzymes catalyzing a short metabolic pathway within a capsid-like shell. BMCs enable metabolism incompatible with their host and this functional advantage is borne out in their pervasiveness. 20-30% of bacterial genomes possess BMC-like proteins. Despite this prevalence, only a handful of BMCs are characterized. One of the most intriguing open questions surrounding BMCs is how a mature functional complex emerges from only protein-protein interactions. Specifically, the mechanism of assembly, cargo ordering and stoichiometry, and the robustness, shape, and size of the mature complex cannot be explained from the current qualitative knowledge of known protein interactions. The goal of our work is to use mechanistic biochemical approaches in order to understand the in vivo self-assembly and function of the BMC known as the α-carboxysome (α-CB). The α-CB facilitates autotrophic growth in many bacteria and was the first BMC to be characterized due to its robustness and ease of biochemical analysis. It is therefore an excellent model system to answer these open questions. Preliminary data indicates that a protein known as CsoS2 is essential for α-CB formation and may be the hub of an interaction network driving self-assembly. We propose to use biochemical and biophysical tools in order to both map the molecular determinants of these interactions and quantitatively understand how multivalency controls assembly. CsoS2 is also an intrinsically disordered protein and possesses numerous repetitive sequence elements. Preliminary data indicates these regions of CsoS2 play an important role in determining α-CB size. Intrinsically disordered proteins are known to participate in an organizing role in eukaryotes, but are largely uncharacterized in prokaryotes. We therefore propose a series of experiments to understand the significance of disorder to CsoS2 function and how its repetitive elements are involved in determining the outcome of the assembly process. Finally, it has long been postulated that BMCs act like an organelle and possess a chemical environment that is distinct from the cytosol. This hypothesis is supported by circumstantial data but has never been directly measured biochemically due to experimental challenges. Here we proposed a series of experiments to make this measurement ex vivo by determining whether the α-CB naturally possesses an oxidative lumen due to the action of its protein shell. We will additionally determine to what extent the chemistry of the lumen affects the self-assembly process. If successful, these experiments will provide novel mechanistic insight into how BMCs assemble and function, and more broadly, the interplay between bacterial ultrastructure and bacterial physiology.