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中文摘要
翻译
细菌细胞具有比通常认为的更多的超微结构组织。一个
英文摘要
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.
期刊论文(4)
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DOI: 10.1021/acs.jpclett.2c00782
发表时间: 2022-05-26
期刊: JOURNAL OF PHYSICAL CHEMISTRY LETTERS
影响因子: 5.7
作者: [Carpenter, William B., Lavania, Abhijit A., Borden, Julia S., Oltrogge, Luke M., Perez, Davis, Dahlberg, Peter D., Savage, David F., Moerner, W. E.]
通讯作者: Moerner, W. E.
DOI: 10.1016/j.mib.2021.03.002
发表时间: 2021-06
期刊: Current opinion in microbiology
影响因子: 5.4
作者: [Borden JS, Savage DF]
通讯作者: Savage DF
Learning to Build a β-Carboxysome.
学习构建β-羧基体。
DOI: 10.1021/acs.biochem.9b00199
发表时间: 2019
期刊: Biochemistry
影响因子: 2.9
作者: [Blikstad,Cecilia, Flamholz,AviI, Oltrogge,LukeM, Savage,DavidF]
通讯作者: Savage,DavidF
DOI: 10.7554/elife.74358
发表时间: 2021-11-09
期刊: eLife
影响因子: 7.7
作者: [Lien KA, Dinshaw K, Nichols RJ, Cassidy-Amstutz C, Knight M, Singh R, Eltis LD, Savage DF, Stanley SA]
通讯作者: Stanley SA
Engineering CRISPR-Cas proteins for conditional and robust interrogation of the genome
  • 批准号:
    9908106
  • 项目类别:
  • 资助金额:
    $30.5万
  • 财政年份:
    2019
  • 负责人:
    David Frank Savage
  • 依托单位:
Engineering CRISPR-Cas proteins for conditional and robust interrogation of the genome
  • 批准号:
    10333376
  • 项目类别:
  • 资助金额:
    $30.4万
  • 财政年份:
    2019
  • 负责人:
    David Frank Savage
  • 依托单位:
Fluorescent biosensors for metabolite imaging in live cells
  • 批准号:
    8571836
  • 项目类别:
  • 资助金额:
    $218.55万
  • 财政年份:
    2013
  • 负责人:
    David Frank Savage
  • 依托单位:
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