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Pore structure controls stability and molecular flux in engineered protein cages

Lachlan S. R. Adamson, Nuren Tasneem, Michael P. Andreas, William Close, Eric N. Jenner, Taylor N. Szyszka, Reginald Young, Li Chen Cheah, Alexander Norman, Hugo I. MacDermott-Opeskin, View ORCID ProfileMegan L. O’Mara, View ORCID ProfileFrank Sainsbury, View ORCID ProfileTobias W. Giessen, View ORCID ProfileYu Heng Lau
doi: https://doi.org/10.1101/2021.01.27.428512
Lachlan S. R. Adamson
1School of Chemistry, The University of Sydney, NSW 2006, Australia
8CSIRO Future Science Platform in Synthetic Biology, Commonwealth Scientific and Industrial Research Organisation (CSIRO), 41 Boggo Road, QLD 4102, Australia
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Nuren Tasneem
1School of Chemistry, The University of Sydney, NSW 2006, Australia
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Michael P. Andreas
2Department of Biomedical Engineering, University of Michigan Medical School, Ann Arbor, MI, USA
3Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA
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William Close
4Australian Centre for Microscopy & Microanalysis, The University of Sydney, NSW 2006, Australia
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Eric N. Jenner
1School of Chemistry, The University of Sydney, NSW 2006, Australia
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Taylor N. Szyszka
1School of Chemistry, The University of Sydney, NSW 2006, Australia
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Reginald Young
1School of Chemistry, The University of Sydney, NSW 2006, Australia
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Li Chen Cheah
6Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, QLD 4072, Australia
8CSIRO Future Science Platform in Synthetic Biology, Commonwealth Scientific and Industrial Research Organisation (CSIRO), 41 Boggo Road, QLD 4102, Australia
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Alexander Norman
1School of Chemistry, The University of Sydney, NSW 2006, Australia
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Hugo I. MacDermott-Opeskin
9Research School of Chemistry, The Australian National University, Canberra, ACT 2601, Australia
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Megan L. O’Mara
9Research School of Chemistry, The Australian National University, Canberra, ACT 2601, Australia
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Frank Sainsbury
6Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, QLD 4072, Australia
7Centre for Cell Factories and Biopolymers, Griffith Institute for Drug Discovery, Griffith University, QLD 4111, Australia
8CSIRO Future Science Platform in Synthetic Biology, Commonwealth Scientific and Industrial Research Organisation (CSIRO), 41 Boggo Road, QLD 4102, Australia
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Tobias W. Giessen
2Department of Biomedical Engineering, University of Michigan Medical School, Ann Arbor, MI, USA
3Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA
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  • For correspondence: yuheng.lau@sydney.edu.au
Yu Heng Lau
1School of Chemistry, The University of Sydney, NSW 2006, Australia
5The University of Sydney Nano Institute, The University of Sydney, NSW 2006, Australia
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  • For correspondence: yuheng.lau@sydney.edu.au
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Abstract

Protein cages are a common architectural motif used by living organisms to compartmentalize and control biochemical reactions. While engineered protein cages have recently been featured in the construction of nanoreactors and synthetic organelles, relatively little is known about the underlying molecular parameters that govern cage stability and molecular flux through their pores. In this work, we systematically designed a 24-member library of protein cage variants based on the T. maritima encapsulin, each featuring pores of different size and charge. Twelve encapsulin pore variants were successfully assembled and purified, including eight designs with exceptional and prolonged thermal stability. While pores lined with negatively charged residues resulted in more robust assemblies than their corresponding positively charged variants, we were able to form stable assemblies covering a full range of pore sizes and charges, as observed in seven new cryo-EM structures of pore variants elucidated at resolutions between 2.5-3.6 Å. Alongside these structures, molecular dynamics simulations and stopped flow kinetics experiments reveal the importance of considering both pore size and surface charge, together with flexibility and rate determining steps, when designing protein cages for controlling molecular flux.

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Competing Interest Statement

The authors have declared no competing interest.

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  • ↵† co-first authors

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Posted August 23, 2021.
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Pore structure controls stability and molecular flux in engineered protein cages
Lachlan S. R. Adamson, Nuren Tasneem, Michael P. Andreas, William Close, Eric N. Jenner, Taylor N. Szyszka, Reginald Young, Li Chen Cheah, Alexander Norman, Hugo I. MacDermott-Opeskin, Megan L. O’Mara, Frank Sainsbury, Tobias W. Giessen, Yu Heng Lau
bioRxiv 2021.01.27.428512; doi: https://doi.org/10.1101/2021.01.27.428512
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Pore structure controls stability and molecular flux in engineered protein cages
Lachlan S. R. Adamson, Nuren Tasneem, Michael P. Andreas, William Close, Eric N. Jenner, Taylor N. Szyszka, Reginald Young, Li Chen Cheah, Alexander Norman, Hugo I. MacDermott-Opeskin, Megan L. O’Mara, Frank Sainsbury, Tobias W. Giessen, Yu Heng Lau
bioRxiv 2021.01.27.428512; doi: https://doi.org/10.1101/2021.01.27.428512

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