Symmetrical protein oligomers are ubiquitous in biological systems and perform key structural and regulatory functions. However, there are few methods for constructing such oligomers. Here we have engineered completely synthetic, symmetrical oligomers by combining pairs of oppositely supercharged variants of a normally monomeric model protein through a strategy we term ‘supercharged protein assembly’ (SuPrA). We show that supercharged variants of green fluorescent protein can assemble into a variety of architectures including a well-defined symmetrical 16-mer structure that we solved using cryo-electron microscopy at 3.47 Å resolution. The 16-mer is composed of two stacked rings of octamers, in which the octamers contain supercharged proteins of alternating charges, and interactions within and between the rings are mediated by a variety of specific electrostatic contacts. The ready assembly of this structure suggests that combining oppositely supercharged pairs of protein variants may provide broad opportunities for generating novel architectures via otherwise unprogrammed interactions.

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Code availability

Source code for HOOMD-blue is available at http://glotzerlab.engin.umich.edu/hoomd-blue/ and at https://bitbucket.org/glotzer/hoomd-blue/. Specific source codes are available upon request.

Data availability

The data generated and analysed in this study, including sequence verification files and the data associated with all figures, are available from the corresponding authors upon reasonable request. The cryo-EM density map of the Ceru+32/GFP−17 protomer has been deposited in the EMDB under accession code EMD-9104. The corresponding atomic model has been deposited in the PDB under accession code 6MDR.

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This material is based on work supported by the US Army Research Laboratory and the US Army Research Office under grant no. W911NF-1–51–0120 to the University of Texas at Austin and under grant no. W911NF-15–1–0185 to the University of Michigan. Computational resources and services for simulation work were supported by Advanced Research Computing at the University of Michigan, Ann Arbor. This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant no. ACI-1053575 (XSEDE award DMR 140129). A.J.S. is supported by an Arnold O. Beckman Postdoctoral Fellowship. D.W.T. is a CPRIT Scholar supported by the Cancer Prevention and Research Institute of Texas (RR160088). This work was supported in part by a Welch Foundation grant F-1938 (to D.W.T.). The authors thank N. Wang for help with building the atomic model, B. Dear for helpful discussions regarding interpretation of DLS data, Texas Materials Institute, part of the Material Science Engineering programme at University Texas at Austin, for supporting the management of the DLS, A. Miklos for helpful discussions regarding supercharged proteins and A. Webb for assistance with confocal microscopy, and the Center for Biomedical Research Support Microscopy and Imaging Facility at the University Texas at Austin for supporting the management of the confocal microscope.

Author information

Author notes

    • Cheulhee Jung

    Present address: Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea

  1. These authors contributed equally: Anna J. Simon and Yi Zhou.


  1. Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, USA

    • Anna J. Simon
    • , Arti Pothukuchy
    • , Barrett R. Morrow
    • , Cheulhee Jung
    • , David W. Taylor
    •  & Andrew D. Ellington
  2. Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA

    • Yi Zhou
    • , Jimmy Gollihar
    • , Jillian C. Gerberich
    • , Janelle C. Leggere
    • , David W. Taylor
    •  & Andrew D. Ellington
  3. Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA

    • Yi Zhou
    • , David W. Taylor
    •  & Andrew D. Ellington
  4. Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, USA

    • Vyas Ramasubramani
    • , Jens Glaser
    •  & Sharon C. Glotzer
  5. US Army Research Laboratory – South, Austin, TX, USA

    • Jimmy Gollihar
  6. Department of Materials Science & Engineering, University of Michigan, Ann Arbor, MI, USA

    • Sharon C. Glotzer
  7. Biointerfaces Institute, University of Michigan, Ann Arbor, MI, USA

    • Sharon C. Glotzer


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A.J.S., Y.Z., V.R., J.Gl., J.Go., A.P., C.J., D.W.T., S.C.G. and A.D.E. conceived and designed the experiments. A.P., A.J.S. and J.Go. designed proteins. A.J.S., B.R.M. and A.P. expressed the proteins. A.P., J.Go. and C.J. performed early optimization of DLS and FRET experiments. A.J.S. and B.R.M. carried out DLS and FRET experiments and analysed the data. J.C.G., J.C.L. and D.W.T. performed the negative stain EM experiments and analysed the data. Y.Z. performed the cryo-EM experiments and atomic model building. A.J.S., Y.Z. and D.W.T. interpreted the cryo-electron microscopy structure and produced the structure figures. V.R. and J.Gl. designed the simulations. V.R. performed simulations. A.J.S. and J.Go. performed and interpreted the confocal microscopy experiments. A.J.S., Y.Z., V.R., J.Gl., S.C.G., D.W.T. and A.D.E. wrote the manuscript, and all authors reviewed and commented on the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to David W. Taylor or Andrew D. Ellington.

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