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De novo engineering of intracellular condensates using artificial disordered proteins

Nature Chemistry volume 12, pages 814–825 (2020)Cite this article

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Abstract

Phase separation of intrinsically disordered proteins (IDPs) is a remarkable feature of living cells to dynamically control intracellular partitioning. Despite the numerous new IDPs that have been identified, progress towards rational engineering in cells has been limited. To address this limitation, we systematically scanned the sequence space of native IDPs and designed artificial IDPs (A-IDPs) with different molecular weights and aromatic content, which exhibit variable condensate saturation concentrations and temperature cloud points in vitro and in cells. We created A-IDP puncta using these simple principles, which are capable of sequestering an enzyme and whose catalytic efficiency can be manipulated by the molecular weight of the A-IDP. These results provide a robust engineered platform for creating puncta with new, phase-separation-mediated control of biological function in living cells.

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Fig. 1: Artificial intrinsically disordered polypeptides inspired from native IDPs exhibit reversible UCST phase behaviour.
Fig. 2: Control of the UCST cloud point using main-chain amino acid composition.
Fig. 3: Control of UCST cloud point by molecular weight of A-IDP.
Fig. 4: A-IDPs exhibit tunable intracellular droplet formation on the basis of the molecular weight and ratio of aromatic to aliphatic content.
Fig. 5: A-IDPs exhibit reversible coacervation in E. coli determined by their molecular weight and aromatic:aliphatic ratio.
Fig. 6: Engineered intracellular droplets with programmable function.
Fig. 7: Engineered intracellular droplets with programmable function.

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Data availability

The data that support the findings of this study are available within this Article and its Supplementary Information. Material requests should be made to chilkoti@duke.edu.

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Acknowledgements

A.C. acknowledges support from the National Institutes of Health (NIH) through an MIRA grant R35GM127042 and from the National Science Foundation (NSF) through a DMREF grant DMR-17-29671. P.S.C acknowledges support from the National Science Foundation through CHE-1709735.

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Authors and Affiliations

  1. Department of Biomedical Engineering, Duke University, Durham, NC, USA

    Michael Dzuricky & Ashutosh Chilkoti

  2. Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, USA

    Bradley A. Rogers & Paul S. Cremer

  3. Department of Computer Science, Duke University, Durham, NC, USA

    Abdulla Shahid

  4. Department of Biology, Duke University, Durham, NC, USA

    Abdulla Shahid

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  1. Michael Dzuricky
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  2. Bradley A. Rogers
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Contributions

M.D. and A.C. designed the experiments and wrote the manuscript. M.D. performed each measurement and analysed the data with the exception of microfluidic temperature-gradient experiments that were performed and analysed by B.A.R. A.S. assisted with UCST cloud-point determination and non-repetitive A-IDP design. P.S.C. assisted in writing the manuscript. All authors discussed the result and commented on the manuscript.

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Correspondence to Ashutosh Chilkoti.

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Extended Materials and Methods, Supplementary Figs. 1–24 and Tables 1–6.

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Dzuricky, M., Rogers, B.A., Shahid, A. et al. De novo engineering of intracellular condensates using artificial disordered proteins. Nat. Chem. 12, 814–825 (2020). https://doi.org/10.1038/s41557-020-0511-7

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