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A structure-based assembly screen of protein cage libraries in living cells: Experimentally repacking a protein-protein interface to recover cage formation in an assembly-frustrated mutant

A structure-based assembly screen of protein cage libraries in living cells: Experimentally repacking a protein-protein interface to recover cage formation in an assembly-frustrated mutant
A structure-based assembly screen of protein cage libraries in living cells: Experimentally repacking a protein-protein interface to recover cage formation in an assembly-frustrated mutant
Cage proteins, which assemble into often highly symmetric hollow nanoscale capsules, have great potential in applications as far reaching as drug delivery, hybrid nanomaterial engineering, and catalysis. In addition, they are promising model systems for understanding how cellular nanostructures are constructed through protein-protein interactions, and they are beginning to be used as scaffolds for synthetic biology approaches. Recently, there has been renewed interest in the engineering of protein cages, and in support of these strategies, we have recently described a fluorescence-based assay for protein cage assembly that is specific for certain oligomerization states and symmetry-related protein-protein interfaces. In this work, we expand this assay to living cells and a high-throughput assay for screening protein cage libraries using flow cytometry. As a proof of principle, we apply this technique to the screening of libraries of a double-alanine mutant of the mini-ferritin, DNA-binding protein from starved cells (Dps). This mutant, due to disruption of key protein-protein interactions, is unable to assemble into a cage. Randomization of residues surrounding the double mutation afforded a repacked interface and proteins with recovered cage formation, demonstrating the strength and utility of this approach.
0006-2960
604-613
Cornell, Thomas A.
267ba1ee-7a4c-4cdb-a01e-0fe9613cbf07
Ardejani, Maziar S.
13136069-41c7-4f67-bf1e-d35dd2968402
Fu, Jing
c4f84e2d-f4f5-49a1-bcb2-8ac1e19693eb
Newland, Stephanie H.
dc459841-89d5-4b96-b862-526b03f30e03
Zhang, Yu
a851ce00-2181-4b85-bb62-50fcbee83a95
Orner, Brendan P.
aebde604-d369-4546-a43c-f1fb13190b3b
Cornell, Thomas A.
267ba1ee-7a4c-4cdb-a01e-0fe9613cbf07
Ardejani, Maziar S.
13136069-41c7-4f67-bf1e-d35dd2968402
Fu, Jing
c4f84e2d-f4f5-49a1-bcb2-8ac1e19693eb
Newland, Stephanie H.
dc459841-89d5-4b96-b862-526b03f30e03
Zhang, Yu
a851ce00-2181-4b85-bb62-50fcbee83a95
Orner, Brendan P.
aebde604-d369-4546-a43c-f1fb13190b3b

Cornell, Thomas A., Ardejani, Maziar S., Fu, Jing, Newland, Stephanie H., Zhang, Yu and Orner, Brendan P. (2018) A structure-based assembly screen of protein cage libraries in living cells: Experimentally repacking a protein-protein interface to recover cage formation in an assembly-frustrated mutant. Biochemistry, 57 (5), 604-613. (doi:10.1021/acs.biochem.7b01000).

Record type: Article

Abstract

Cage proteins, which assemble into often highly symmetric hollow nanoscale capsules, have great potential in applications as far reaching as drug delivery, hybrid nanomaterial engineering, and catalysis. In addition, they are promising model systems for understanding how cellular nanostructures are constructed through protein-protein interactions, and they are beginning to be used as scaffolds for synthetic biology approaches. Recently, there has been renewed interest in the engineering of protein cages, and in support of these strategies, we have recently described a fluorescence-based assay for protein cage assembly that is specific for certain oligomerization states and symmetry-related protein-protein interfaces. In this work, we expand this assay to living cells and a high-throughput assay for screening protein cage libraries using flow cytometry. As a proof of principle, we apply this technique to the screening of libraries of a double-alanine mutant of the mini-ferritin, DNA-binding protein from starved cells (Dps). This mutant, due to disruption of key protein-protein interactions, is unable to assemble into a cage. Randomization of residues surrounding the double mutation afforded a repacked interface and proteins with recovered cage formation, demonstrating the strength and utility of this approach.

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More information

e-pub ahead of print date: 25 December 2017
Published date: 6 February 2018

Identifiers

Local EPrints ID: 419813
URI: https://eprints.soton.ac.uk/id/eprint/419813
ISSN: 0006-2960
PURE UUID: 7dc8840e-95c1-4e30-a146-ac617e74117b

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Date deposited: 20 Apr 2018 16:30
Last modified: 16 Jan 2019 17:32

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Contributors

Author: Thomas A. Cornell
Author: Maziar S. Ardejani
Author: Jing Fu
Author: Stephanie H. Newland
Author: Yu Zhang
Author: Brendan P. Orner

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