The self-assembly of bacterial microcompartments is the result of several genetic, biochemical, and physical stimuli orchestrating inside the bacterial cell. In this work, we use 1,2-propanediol utilization microcompartments as a paradigm to identify the factors that physically drive the self-assembly of MCP proteins in vitro using its major shell protein and major encapsulated enzyme. We find that a major shell protein PduBB′ tends to self-assemble under macromolecular crowded environment and suitable ionic strength. Microscopic visualization and biophysical studies reveal phase separation to be the principle mechanism behind the self-association of shell protein in the presence of salts and macromolecular crowding. The shell protein PduBB′ interacts with the enzyme diol-dehydratase PduCDE and co-assemble into phase separated liquid droplets. The co-assembly of PduCDE and PduBB′ results in the enhancement of catalytic activity of the enzyme. The shell proteins that make up PduBB′ (PduB and PduB′) have contrasting self-assembly behavior. While N-terminal truncated PduB′ has a high self-associating property and forms solid assemblies that separates out of solution, the longer component of the shell protein PduBM38L is more soluble and shows least tendency to undergo phase separation. A combination of spectroscopic, imaging and biochemical techniques shows the relevance of divalent cation Mg2+ in providing stability to intact PduMCP. Together our results suggest a combination of protein–protein interactions and phase separation guiding the self-assembly of Pdu shell protein and enzyme in the solution phase.
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April 2023
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Cover Image
The cover image is part of a larger image demonstrating that shell protein PduBBʹ has higher LLPS propensity than enzyme PduCDE and shows the time dependent recruitment of Texas red-labelled PduCDE within Alexa-488 labelled phase separated PduBBʹ. For more information see the article by Kumar and Sinha (pp. 539–553) in this issue. The image is courtesy of Sharmistha Sinha.
Research Article|
April 26 2023
Self-assembly of shell protein and native enzyme in a crowded environment leads to catalytically active phase condensates
Gaurav Kumar;
Gaurav Kumar
Data curation, Formal analysis, Writing - original draft
Chemical Biology Unit, Institute of Nano Science and Technology, Knowledge City, Sector-81, Mohali 140306, India
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Sharmistha Sinha
Chemical Biology Unit, Institute of Nano Science and Technology, Knowledge City, Sector-81, Mohali 140306, India
Correspondence: Sharmistha Sinha (sinhas@inst.ac.in)
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Publisher: Portland Press Ltd
Received:
November 08 2022
Revision Received:
January 20 2023
Accepted:
January 20 2023
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society
2023
Biochem J (2023) 480 (8): 539–553.
Article history
Received:
November 08 2022
Revision Received:
January 20 2023
Accepted:
January 20 2023
Citation
Gaurav Kumar, Sharmistha Sinha; Self-assembly of shell protein and native enzyme in a crowded environment leads to catalytically active phase condensates. Biochem J 26 April 2023; 480 (8): 539–553. doi: https://doi.org/10.1042/BCJ20220551
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