Assembling membraneless organelles from de novo designed proteins

Alexander T. Hilditch; Andrey Romanyuk; Stephen J. Cross; Richard Obexer; Jennifer J. McManus; Derek N. Woolfson

doi:10.1101/2023.04.18.537322

Assembling membraneless organelles from de novo designed proteins

Alexander T. Hilditch, Andrey Romanyuk, Stephen J. Cross, Richard Obexer^*, Jennifer J. McManus^*, Derek N. Woolfson^*

^*Corresponding author for this work

Research output: Contribution to journal › Article (Academic Journal) › peer-review

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Abstract

Recent advances in de novo protein design have delivered a diversity of discrete de novo protein structures and complexes. A new challenge for the field is to use these designs directly in cells to intervene in biological processes and augment natural systems. The bottom-up design of self-assembled objects such as microcompartments and membraneless organelles is one such challenge. Here we describe the design of genetically encoded polypeptides that form membraneless organelles in Escherichia coli. To do this, we combine de novo α-helical sequences, intrinsically disordered linkers and client proteins in single-polypeptide constructs. We tailor the properties of the helical regions to shift protein assembly from arrested assemblies to dynamic condensates. The designs are characterized in cells and in vitro using biophysical methods and soft-matter physics. Finally, we use the designed polypeptide to co-compartmentalize a functional enzyme pair in E. coli, improving product formation close to the theoretical limit. [Figure not available: see fulltext.].

Original language	English
Pages (from-to)	89-97
Number of pages	9
Journal	Nature Chemistry
Volume	16
Issue number	1
Early online date	14 Sept 2023
DOIs	https://doi.org/10.1101/2023.04.18.537322 https://doi.org/10.1038/s41557-023-01321-y
Publication status	Published - 1 Jan 2024

Bibliographical note

Funding Information:
A.T.H. and D.N.W. are funded by the University of Bristol through the Max Planck-Bristol Centre for Minimal Biology. A.R. is funded by the Leverhulme Trust through a grant to J.J.M. and D.N.W. (RGP-2021-049). R.O. was funded through a European Union’s Horizon 2020 research and innovation programme Marie Skłodowska-Curie grant (NovoFold no. 795867). We thank the University of Bristol School of Chemistry Mass Spectrometry Facility for access to the EPSRC-financed Bruker Ultraflex MALDI-TOF/TOF instrument (EP/K03927X/1), BrisSynBio for access to peptide synthesizers (BB/L01386X/1), and the Wolfson Bioimaging Facility for their assistance in this work. We thank C. Zhang of Tsinghua University for providing a BL21 (DE3) ∆tnaA E. coli strain. We thank M. Lee for the pDIC bacterial vectors. We thank P. Wilson (BioSuite, University of Bristol) for use of the Formulatrix crystallization hotel, and A. Strofaldi for assistance with the soft-matter experiments.

Funding Information:
A.T.H. and D.N.W. are funded by the University of Bristol through the Max Planck-Bristol Centre for Minimal Biology. A.R. is funded by the Leverhulme Trust through a grant to J.J.M. and D.N.W. (RGP-2021-049). R.O. was funded through a European Union’s Horizon 2020 research and innovation programme Marie Skłodowska-Curie grant (NovoFold no. 795867). We thank the University of Bristol School of Chemistry Mass Spectrometry Facility for access to the EPSRC-financed Bruker Ultraflex MALDI-TOF/TOF instrument (EP/K03927X/1), BrisSynBio for access to peptide synthesizers (BB/L01386X/1), and the Wolfson Bioimaging Facility for their assistance in this work. We thank C. Zhang of Tsinghua University for providing a BL21 (DE3) ∆tnaA E. coli strain. We thank M. Lee for the pDIC bacterial vectors. We thank P. Wilson (BioSuite, University of Bristol) for use of the Formulatrix crystallization hotel, and A. Strofaldi for assistance with the soft-matter experiments.

Publisher Copyright:
© 2023, The Author(s).

Structured keywords

Bristol BioDesign Institute
Max Planck Bristol
BrisSynBio

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title = "Assembling membraneless organelles from de novo designed proteins",

abstract = "Recent advances in de novo protein design have delivered a diversity of discrete de novo protein structures and complexes. A new challenge for the field is to use these designs directly in cells to intervene in biological processes and augment natural systems. The bottom-up design of self-assembled objects such as microcompartments and membraneless organelles is one such challenge. Here we describe the design of genetically encoded polypeptides that form membraneless organelles in Escherichia coli. To do this, we combine de novo α-helical sequences, intrinsically disordered linkers and client proteins in single-polypeptide constructs. We tailor the properties of the helical regions to shift protein assembly from arrested assemblies to dynamic condensates. The designs are characterized in cells and in vitro using biophysical methods and soft-matter physics. Finally, we use the designed polypeptide to co-compartmentalize a functional enzyme pair in E. coli, improving product formation close to the theoretical limit. [Figure not available: see fulltext.].",

author = "Hilditch, {Alexander T.} and Andrey Romanyuk and Cross, {Stephen J.} and Richard Obexer and McManus, {Jennifer J.} and Woolfson, {Derek N.}",

note = "Funding Information: A.T.H. and D.N.W. are funded by the University of Bristol through the Max Planck-Bristol Centre for Minimal Biology. A.R. is funded by the Leverhulme Trust through a grant to J.J.M. and D.N.W. (RGP-2021-049). R.O. was funded through a European Union{\textquoteright}s Horizon 2020 research and innovation programme Marie Sk{\l}odowska-Curie grant (NovoFold no. 795867). We thank the University of Bristol School of Chemistry Mass Spectrometry Facility for access to the EPSRC-financed Bruker Ultraflex MALDI-TOF/TOF instrument (EP/K03927X/1), BrisSynBio for access to peptide synthesizers (BB/L01386X/1), and the Wolfson Bioimaging Facility for their assistance in this work. We thank C. Zhang of Tsinghua University for providing a BL21 (DE3) ∆tnaA E. coli strain. We thank M. Lee for the pDIC bacterial vectors. We thank P. Wilson (BioSuite, University of Bristol) for use of the Formulatrix crystallization hotel, and A. Strofaldi for assistance with the soft-matter experiments. Funding Information: A.T.H. and D.N.W. are funded by the University of Bristol through the Max Planck-Bristol Centre for Minimal Biology. A.R. is funded by the Leverhulme Trust through a grant to J.J.M. and D.N.W. (RGP-2021-049). R.O. was funded through a European Union{\textquoteright}s Horizon 2020 research and innovation programme Marie Sk{\l}odowska-Curie grant (NovoFold no. 795867). We thank the University of Bristol School of Chemistry Mass Spectrometry Facility for access to the EPSRC-financed Bruker Ultraflex MALDI-TOF/TOF instrument (EP/K03927X/1), BrisSynBio for access to peptide synthesizers (BB/L01386X/1), and the Wolfson Bioimaging Facility for their assistance in this work. We thank C. Zhang of Tsinghua University for providing a BL21 (DE3) ∆tnaA E. coli strain. We thank M. Lee for the pDIC bacterial vectors. We thank P. Wilson (BioSuite, University of Bristol) for use of the Formulatrix crystallization hotel, and A. Strofaldi for assistance with the soft-matter experiments. Publisher Copyright: {\textcopyright} 2023, The Author(s).",

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AU - Woolfson, Derek N.

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Assembling membraneless organelles from de novo designed proteins

Abstract

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Assembling membraneless organelles from de novo designed proteins

Abstract

Bibliographical note

Structured keywords

Access to Document

Handle.net

Other files and links

Fingerprint

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Data associated with the publication "Assembling membraneless organelles from de novo designed proteins", v1

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