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Supercharging enables organized assembly of synthetic biomolecules

Nature Chemistry volume 11, pages 204–212 (2019)Cite this article

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Abstract

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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Fig. 1: Oppositely supercharged Cerulean (Ceru) and GFP variants as a model system for charge-mediated protein assembly.
Fig. 2: Ceru+32/GFP−17 particle size depends on NaCl concentration.
Fig. 3: Cryo-EM structure of the Ceru+32/GFP−17 protomer.
Fig. 4: Inter-protein interactions in the Ceru+32/GFP−17 protomer.
Fig. 5: Computational simulations of protomer structure stability.
Fig. 6: Confocal images of micrometre-scale Ceru+32/GFP−17 particles.

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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.

References

  1. Whitesides, G. M. & Grzybowski, B. Self-assembly at all scales. Science 295, 2418–2421 (2002).

    Article  CAS  Google Scholar 

  2. Blundell, T. L. & Srinivasan, N. Symmetry, stability, and dynamics of multidomain and multicomponent protein systems. Proc. Natl Acad. Sci. USA 93, 14243–14248 (1996).

    Article  CAS  Google Scholar 

  3. Levy, E. D., Boeri Erba, E., Robinson, C. V. & Teichmann, S. A. Assembly reflects evolution of protein complexes. Nature 453, 1262–1265 (2008).

    Article  CAS  Google Scholar 

  4. Goodsell, D. S. & Olson, A. J. Structural symmetry and protein function. Annu. Rev. Biophys. Biomol. Struct. 29, 105–153 (2000).

    Article  CAS  Google Scholar 

  5. André, I., Strauss, C. E., Kaplan, D. B., Bradley, P. & Baker, D. Emergence of symmetry in homooligomeric biological assemblies. Proc. Natl Acad. Sci. USA 105, 16148–16152 (2008).

    Article  Google Scholar 

  6. Plaxco, K. W. & Gross, M. Protein complexes: the evolution of symmetry. Curr. Biol. 19, R25–R26 (2009).

    Article  CAS  Google Scholar 

  7. Bergendahl, L. T. & Marsh, J. H. Functional determinants of protein assembly into homomeric complexes. Sci. Rep. 7, 4932 (2017).

    Article  Google Scholar 

  8. Padilla, J. E., Colovos, C. & Yeates, T. O. Nanohedra: using symmetry to design self assembling protein cages, layers, crystals and filaments. Proc. Natl Acad. Sci. USA 98, 2217–2221 (2001).

    Article  CAS  Google Scholar 

  9. Lai, Y.-T., Cascio, D. & Yeates, T. O. Structure of a 16-nm cage designed by using protein oligomers. Science 336, 1129 (2012).

    Article  CAS  Google Scholar 

  10. Alberstein, R., Suzuki, Y., Paesani, F. & Tezcan, F. A. Engineering the entropy-driven free-energy landscape of a dynamic nanoporous protein assembly. Nat. Chem. 10, 732–739 (2018).

    Article  CAS  Google Scholar 

  11. Badieyan, S. et al. Symmetry‐directed self‐assembly of a tetrahedral protein cage mediated by de novo‐designed coiled coils. Chembiochem 18, 1888–1892 (2017).

    Article  CAS  Google Scholar 

  12. Brodin, J. D., Carr, J. R., Sontz, P. A. & Tezcan, F. A. Exceptionally stable, redox-active supramolecular protein assemblies with emergent properties. Proc. Natl Acad. Sci. USA 111, 2897–2902 (2014).

    Article  CAS  Google Scholar 

  13. Yeates, T. O. Geometric principles for designing highly symmetric self-assembling protein nanomaterials. Annu. Rev. Biophys. 46, 23–42 (2017).

    Article  CAS  Google Scholar 

  14. Boyken, S. E. et al. De novo design of protein homo-oligomers with modular hydrogen-bond network-mediated specificity. Science 352, 680–687 (2016).

    Article  CAS  Google Scholar 

  15. Bale, J. B. et al. Accurate design of megadalton-scale two-component icosahedral protein complexes. Science 353, 389–394 (2016).

    Article  CAS  Google Scholar 

  16. Hsia, Y. et al. Design of a hyperstable 60-subunit protein dodecahedron. Nature 535, 136–139 (2016).

    Article  CAS  Google Scholar 

  17. Butterfield, G. L. et al. Evolution of a designed protein assembly encapsulating its own RNA genome. Nature 552, 415–420 (2017).

    Article  CAS  Google Scholar 

  18. Kobayashi, N. & Arai, R. Design and construction of self-assembling supramolecular protein complexes using artificial and fusion proteins as nanoscale building blocks. Curr. Opin. Biotechnol. 46, 57–65 (2017).

    Article  CAS  Google Scholar 

  19. Damasceno, P. F., Engel, M. & Glotzer, S. C. Predictive self-assembly of polyhedra into complex structures. Science 337, 453–457 (2012).

    Article  CAS  Google Scholar 

  20. Paik, T. & Murray, C. B. Shape-directed binary assembly of anisotropic nanoplates: a nanocrystal puzzle with shape-complementary building blocks. Nano Lett. 13, 2952–2956 (2013).

    Article  CAS  Google Scholar 

  21. Gong, J. et al. Shape-dependent ordering of gold nanocrystals into large-scale superlattices. Nat. Commun. 8, 14038 (2017).

    Article  CAS  Google Scholar 

  22. Wolters, J. R. et al. Self-assembly of ‘Mickey Mouse’ shaped colloids into tube-like structures: experiments and simulations. Soft Matter 11, 1067–1077 (2015).

    Article  CAS  Google Scholar 

  23. Boles, M. A., Engel, M. & Talapin, D. V. Self-assembly of colloidal nanocrystals: from intricate structures to functional materials. Chem. Rev. 116, 11220–11289 (2016).

    Article  CAS  Google Scholar 

  24. Fu, L. et al. Assembly of hard spheres in a cylinder: a computational and experimental study. Soft Matter 13, 3296–3306 (2017).

    Article  CAS  Google Scholar 

  25. Ye, X. et al. Competition of shape and interaction patchiness for self-assembling nanoplates. Nat. Chem. 5, 466–473 (2013).

    Article  CAS  Google Scholar 

  26. Zhang, Z. & Glotzer, S. C. Self-assembly of patchy particles. Nano Lett. 4, 1407–1413 (2004).

    Article  CAS  Google Scholar 

  27. Giacometti, A., Lado, F., Largo, J., Pastore, G. & Sciortino, F. Effects of patch size and number within a simple model of patchy colloids. J. Chem. Phys. 132, 174110 (2010).

    Article  Google Scholar 

  28. Pawar, A. B. & Kretzschmar, I. Fabrication, assembly and application of patchy particles. Macromol. Rapid Commun. 31, 150–168 (2010).

    Article  CAS  Google Scholar 

  29. Gong, Z., Hueckel, T., Yi, G. R. & Sacanna, S. Patchy particles made by colloidal fusion. Nature 550, 234–238 (2017).

    Article  Google Scholar 

  30. Duguet, E., Hubert, C., Chomette, C., Perroc, A. & Ravaine, S. Patchy colloidal particles for programmed self-assembly. C. R. Chimi 19, 173–182 (2016).

    Article  CAS  Google Scholar 

  31. Woo, S. & Rothemund, P. W. Programmable molecular recognition based on the geometry of DNA nanostructures. Nat. Chem. 3, 620–627 (2011).

    Article  CAS  Google Scholar 

  32. Gerling, T., Wagenbauer, K. F., Neunerm, A. M. & Dietz, H. Dynamic DNA devices and assemblies formed by shape-complementary, non-base pairing 3D components. Science 347, 1446–1452 (2015).

    Article  CAS  Google Scholar 

  33. Sheinerman, F. B., Norel, R. & Honig, B. Electrostatic aspects of protein–protein interactions. Curr. Opin. Struct. Biol. 10, 153–159 (2000).

    Article  CAS  Google Scholar 

  34. Liljeström, V., Mikkilä, J. & Kostiainen, M. A. Self-assembly and modular functionalization of three-dimensional crystals from oppositely charged proteins. Nat. Commun. 5, 4445 (2014).

    Article  Google Scholar 

  35. Kostiainen, M. A. et al. Electrostatic assembly of binary nanoparticle superlattices using protein cages. Nat. Nanotech. 8, 52–56 (2012).

    Article  Google Scholar 

  36. Seebeck, F. P., Woycechowsky, K. J., Zhuang, W., Rabe, J. P. & Hilvert, D. A simple tagging system for protein encapsulation. J. Am. Chem. Soc. 128, 4516–4517 (2006).

    Article  CAS  Google Scholar 

  37. Wörsdörfer, B., Woycechowsky, K. J. & Hilvert, D. Directed evolution of a protein container. Science 331, 589–592 (2011).

    Article  Google Scholar 

  38. Held, M. et al. Engineering formation of multiple recombinant Eut protein nanocompartments in E. coli. Sci. Rep. 6, 24359 (2016).

    Article  CAS  Google Scholar 

  39. Beck, T., Tetter, S., Künzle, M. & Hilvert, D. Construction of Matryoshka-type structures from supercharged protein nanocages. Angew. Chem. Int. Ed. 54, 937–940 (2014).

    Article  Google Scholar 

  40. Sun, H., Luo, Q., Hou, C. & Liu, J. Nanostructures based on protein self-assembly: from hierarchical construction to bioinspired materials. Nanotoday 14, 16–41 (2017).

    Article  CAS  Google Scholar 

  41. Lawrence, M. S., Phillips, K. J. & Liu, D. R. Supercharging proteins can impart unusual resilience. J. Am. Chem. Soc. 129, 10110–10112 (2007).

    Article  CAS  Google Scholar 

  42. Der, B. S. et al. Alternative computational protocols for supercharging protein surfaces for reversible unfolding and retention of stability. PLoS One 31, e64363 (2013).

    Article  Google Scholar 

  43. Oh, H. J., Gather, M. C., Song, J.-J. & Yun, S. H. Lasing from fluorescent protein crystals. Opt. Express 22, 31411–31416 (2014).

    Article  CAS  Google Scholar 

  44. Ormö, M. et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996).

    Article  Google Scholar 

  45. Bolhuis, P. & Frenkel, D. Tracing the phase boundaries of hard spherocylinders. J. Chem. Phys. 106, 666–687 (1997).

    Article  CAS  Google Scholar 

  46. Rizzo, M. A., Springer, G. H., Granada, B. & Piston, D. W. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 4, 445–449 (2004).

    Article  Google Scholar 

  47. Goedhart, J. et al. Bright cyan fluorescent protein variants identified by fluorescence lifetime screening. Nat. Methods 7, 137–139 (2010).

    Article  CAS  Google Scholar 

  48. Goedhart, J. et al. Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nat. Commun. 3, 751 (2012).

    Article  Google Scholar 

  49. García-Seisdedos, H., Empereur-Mot, C., Elad, N. & Levy, E. D. Proteins evolve on the edge of supramolecular self-assembly. Nature 548, 244–247 (2017).

    PubMed  Google Scholar 

  50. Hassan, P. A., Rana, S. & Verma, G. Making sense of Brownian motion: colloid characterization by dynamic light scattering. Langmuir 31, 3–12 (2015).

    Article  CAS  Google Scholar 

  51. Bajar, B. T., Wang, E. S., Zhang, S., Lin, M. Z. & Chu, J. A guide to fluorescent protein FRET pairs. Sensors 16, E1488 (2016).

    Article  Google Scholar 

  52. Anderson, J. A., Lorenz, C. D. & Travesset, A. General purpose molecular dynamics simulations fully implemented on graphics processing units. J. Comp. Phys. 227, 5342–5359 (2008).

    Article  Google Scholar 

  53. Glaser, J. et al. Strong scaling of general-purpose molecular dynamics simulations on GPUs. Comp. Phys. Commun. 192, 97–107 (2015).

    Article  CAS  Google Scholar 

  54. Sinkovits, D. W., Barr, S. A. & Luijten, E. Rejection-free Monte Carlo scheme for anisotropic particles. J. Chem. Phys. 136, 144111 (2012).

    Article  Google Scholar 

  55. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    Article  CAS  Google Scholar 

  56. Pédelacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C. & Waldo, G. S. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 79–88 (2006).

    Article  Google Scholar 

  57. Yang, F., Moss, L. G. & Phillips, G. B. Jr. The molecular structure of green fluorescent protein. Nat. Biotechnol. 14, 1246–1251 (1996).

    Article  CAS  Google Scholar 

  58. Lin, M. Y. et al. Universal diffusion-limited colloid aggregation. J. Phys. Condens. Matter 2, 3039–3113 (1990).

    Article  Google Scholar 

  59. Miklos, A. E. et al. Structure-based design of supercharged, highly thermoresistant antibodies. Chem. Biol. 19, 449–455 (2012).

    Article  CAS  Google Scholar 

  60. Johnson, L. B., Park, S., Gintner, L. P. & Snow, C. D. Characterization of supercharged cellulase activity and stability in ionic liquids. J. Mol. Catal. B 132, 84–90 (2016).

    Article  CAS  Google Scholar 

  61. Zuris, J. A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2015).

    Article  CAS  Google Scholar 

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Acknowledgements

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.

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Author notes

  1. Cheulhee Jung

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

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

Authors and Affiliations

  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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Contributions

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.

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

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Simon, A.J., Zhou, Y., Ramasubramani, V. et al. Supercharging enables organized assembly of synthetic biomolecules. Nature Chem 11, 204–212 (2019). https://doi.org/10.1038/s41557-018-0196-3

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