Self-assembly and function of bacterial microcompartments

细菌细胞具有比通常认识到的显著更多的超微结构组织。一 最引人注目的例子是细菌微区室（BMC），大（即100+ nm） 包封货物酶的蛋白质复合物，催化短代谢途径， 一种像贝壳一样的贝壳。BMC使代谢与其宿主不相容， 在他们的普遍性中得到了证实。20-30%的细菌基因组具有BMC样蛋白。尽管 这种流行，只有少数的BMC的特点。最有趣的开放性问题之一 是一个成熟的功能复合物如何从蛋白质-蛋白质相互作用中出现的。 具体而言，组装机制，货物排序和化学计量，以及鲁棒性，形状， 和规模的成熟复杂的不能解释从目前的定性知识的已知 蛋白质相互作用我们工作的目标是使用机械生物化学方法， 了解称为α-羧基体（α-CB）的BMC的体内自组装和功能。的 α-CB促进许多细菌的自养生长，是第一个被表征的BMC，因为它具有 生化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。 开放性问题。初步数据表明，一种称为CsoS 2的蛋白质对α-CB的形成至关重要 并且可以是驱动自组装的交互网络的中心。我们建议使用生物化学和 生物物理工具，以便既映射这些相互作用的分子决定因素， 了解多价性如何控制组装。CsoS 2也是一种内在无序的蛋白质， 具有许多重复序列元件。初步数据表明CsoS 2的这些区域 在确定α-CB尺寸中起重要作用。已知胞内无序蛋白参与 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 一系列的实验来了解紊乱对CsoS 2功能的意义以及其重复性如何影响CsoS 2的功能。 在决定大会进程的结果时涉及到各种因素。最后， 假设BMC像细胞器一样起作用，并且具有与细胞器不同的化学环境。 胞质液这一假设得到了间接数据的支持，但从未得到直接测量 生物化学的挑战。在这里我们提出了一系列的实验， 通过测定α-CB是否天然具有氧化腔（由于 蛋白质外壳的作用。我们还将确定管腔的化学性质 影响了自组装过程。如果成功，这些实验将提供新的机制见解 了解BMC如何组装和发挥功能，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.