Programming molecular self-assembly of intrinsically disordered proteins containing sequences of low complexity

Abstract

Dynamic protein-rich intracellular structures that contain phase-separated intrinsically disordered proteins (IDPs) composed of sequences of low complexity (SLC) have been shown to serve a variety of important cellular functions, which include signalling, compartmentalization and stabilization. However, our understanding of these structures and our ability to synthesize models of them have been limited. We present design rules for IDPs possessing SLCs that phase separate into diverse assemblies within droplet microenvironments. Using theoretical analyses, we interpret the phase behaviour of archetypal IDP sequences and demonstrate the rational design of a vast library of multicomponent protein-rich structures that ranges from uniform nano-, meso- and microscale puncta (distinct protein droplets) to multilayered orthogonally phase-separated granular structures. The ability to predict and program IDP-rich assemblies in this fashion offers new insights into (1) genetic-to-molecular-to-macroscale relationships that encode hierarchical IDP assemblies, (2) design rules of such assemblies in cell biology and (3) molecular-level engineering of self-assembled recombinant IDP-rich materials.

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Figure 1: Programming of artificial liquid coacervates from ELPs.
Figure 2: Reversible formation of ELP coacervates inside droplets by spinodal decomposition and resolubilzation.
Figure 3: Multicomponent solutions of ELPs enable the formation of layered and mixed coacervates.
Figure 4: Amphiphilic proteins kinetically arrest coalescence of ELPs during phase separation to produce uniform nano-, meso- and microscale puncta.

References

  1. 1

    Rubinstein, M. & Colby, R. Polymers Physics (Oxford Univ. Press, 2003).

    Google Scholar 

  2. 2

    Long, M. S., Jones, C. D., Helfrich, M. R., Mangeney-Slavin, L. K. & Keating, C. D. Dynamic microcompartmentation in synthetic cells. Proc. Natl Acad. Sci. USA 102, 5920–5925 (2005).

    CAS  Article  Google Scholar 

  3. 3

    Li, P. et al. Phase transitions in the assembly of multivalent signalling proteins. Nature 483, 336–340 (2012).

    CAS  Article  Google Scholar 

  4. 4

    Luby-Phelps, K. The physical chemistry of cytoplasm and its influence on cell function: an update. Mol. Biol. Cell 24, 2593–2596 (2013).

    CAS  Article  Google Scholar 

  5. 5

    Hyman, A. A. & Simons, K. Beyond oil and water—phase transitions in cells. Science 337, 1047–1049 (2012).

    CAS  Article  Google Scholar 

  6. 6

    Nott, T. J. et al. Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles. Mol. Cell 57, 936–947 (2015).

    CAS  Article  Google Scholar 

  7. 7

    Han, T. W. et al. Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies. Cell 149, 768–779 (2012).

    CAS  Article  Google Scholar 

  8. 8

    Kato, M. et al. Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Cell 149, 753–767 (2012).

    CAS  Article  Google Scholar 

  9. 9

    Weber, S. C. & Brangwynne, C. P. Getting RNA and protein in phase. Cell 149, 1188–1191 (2012).

    CAS  Article  Google Scholar 

  10. 10

    Parker, R. & Sheth, U. P bodies and the control of mRNA translation and degradation. Mol. Cell 25, 635–646 (2007).

    CAS  Article  Google Scholar 

  11. 11

    Bernardi, R. & Pandolfi, P. P. Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies. Nat. Rev. Mol. Cell Biol. 8, 1006–1016 (2007).

    CAS  Article  Google Scholar 

  12. 12

    An, S., Kumar, R., Sheets, E. D. & Benkovic, S. J. Reversible compartmentalization of de novo purine biosynthetic complexes in living cells. Science 320, 103–106 (2008).

    CAS  Article  Google Scholar 

  13. 13

    Elbaum-Garfinkle, S. et al. The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics. Proc. Natl Acad. Sci. USA 112, 7189–7194 (2015).

    CAS  Article  Google Scholar 

  14. 14

    Nott, T. J., Craggs, T. D. & Baldwin, A. J. Membraneless organelles can melt nucleic acid duplexes and act as biomolecular filters. Nat. Chem. 8, 569–575 (2016).

    CAS  Article  Google Scholar 

  15. 15

    Roberts, S., Dzuricky, M. & Chilkoti, A. Elastin-like polypeptides as models of intrinsically disordered proteins. FEBS Lett. 589, 2477–2486 (2015).

    CAS  Article  Google Scholar 

  16. 16

    Meyer, D. E. & Chilkoti, A. Purification of recombinant proteins by fusion with thermally responsive polypeptides. Nat. Biotech. 17, 1112–1115 (1999).

    CAS  Article  Google Scholar 

  17. 17

    Urry, D. W. Physical chemistry of biological free energy transduction as demonstrated by elastic protein-based polymers. J. Phys. Chem. B 101, 11007–11028 (1997).

    CAS  Article  Google Scholar 

  18. 18

    McDaniel, J. R., Radford, D. C. & Chilkoti, A. A unified model for de novo design of elastin-like polypeptides with tunable inverse transition temperatures. Biomacromolecules 14, 2866–2872 (2013).

    CAS  Article  Google Scholar 

  19. 19

    Pak, C. W. et al. Sequence determinants of intracellular phase separation by complex coacervation of a disordered protein. Mol. Cell 63, 72–85 (2016).

    CAS  Article  Google Scholar 

  20. 20

    Meyer, D. E. & Chilkoti, A. Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system. Biomacromolecules 3, 357–367 (2002).

    CAS  Article  Google Scholar 

  21. 21

    Meyer, D. E. & Chilkoti, A. Quantification of the effects of chain length and concentration on the thermal behavior of elastin-like polypeptides. Biomacromolecules 5, 846–851 (2004).

    CAS  Article  Google Scholar 

  22. 22

    Dreher, M. R. et al. Temperature triggered self-assembly of polypeptides into multivalent spherical micelles. J. Am. Chem. Soc. 130, 687–694 (2008).

    CAS  Article  Google Scholar 

  23. 23

    Cho, Y. et al. Effects of Hofmeister anions on the phase transition temperature of elastin-like polypeptides. J. Phys. Chem. B 112, 13765–13771 (2008).

    CAS  Article  Google Scholar 

  24. 24

    Hassouneh, W., Zhulina, E. B., Chilkoti, A. & Rubinstein, M. Elastin-like polypeptide diblock copolymers self-assemble into weak micelles. Macromolecules 48, 4183–4195 (2015).

    CAS  Article  Google Scholar 

  25. 25

    Mart, R. J., Osborne, R. D., Stevens, M. M. & Ulijn, R. V. Peptide-based stimuli-responsive biomaterials. Soft Matter 2, 822–835 (2006).

    CAS  Article  Google Scholar 

  26. 26

    Cahn, J. W. & Hilliard, J. E. Free energy of a nonuniform system. I. Interfacial free energy. J. Chem. Phys. 28, 258–267 (1958).

    CAS  Article  Google Scholar 

  27. 27

    Nesterov, A. E. & Lipatov, Y. S. Thermodynamics of Polymer Blends Vol. 1 (CRC, 1998).

    Google Scholar 

  28. 28

    Brangwynne, C. P., Mitchison, T. J. & Hyman, A. A. Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes. Proc. Natl Acad. Sci. USA 108, 4334–4339 (2011).

    CAS  Article  Google Scholar 

  29. 29

    Petsev, D. N., Wu, X., Galkin, O. & Vekilov, P. G. Thermodynamic functions of concentrated protein solutions from phase equilibria. J. Phys. Chem. B 107, 3921–3926 (2003).

    CAS  Article  Google Scholar 

  30. 30

    Schmidt, H. B. & Rohatgi, R. In vivo formation of vacuolated multi-phase compartments lacking membranes. Cell Rep. 16, 1228–1236 (2016).

    CAS  Article  Google Scholar 

  31. 31

    Feric, M. et al. Coexisting liquid phases underlie nucleolar subcompartments. Cell 165, 1686–1697 (2016).

    CAS  Article  Google Scholar 

  32. 32

    Jain, S. et al. ATPase-modulated stress granules contain a diverse proteome and substructure. Cell 164, 487–498 (2016).

    CAS  Article  Google Scholar 

  33. 33

    Izzo, D. & Marques, C. M. Solubilization of homopolymers in a solution of diblock copolymers. J. Phys. Chem. B 109, 6140–6145 (2005).

    CAS  Article  Google Scholar 

  34. 34

    Gall, J. G., Bellini, M., Wu, Z. A. & Murphy, C. Assembly of the nuclear transcription and processing machinery: Cajal bodies (coiled bodies) and transcriptosomes. Mol. Biol. Cell 10, 4385–4402 (1999).

    CAS  Article  Google Scholar 

  35. 35

    McDaniel, J. R., MacKay, J. A., Quiroz, F. G. A. & Chilkoti, A. Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes. Biomacromolecules 11, 944–952 (2010).

    CAS  Article  Google Scholar 

  36. 36

    Thomson, J. A., Schurtenberger, P., Thurston, G. M. & Benedek, G. B. Binary liquid phase separation and critical phenomena in a protein/water solution. Proc. Natl Acad. Sci. USA 84, 7079–7083 (1987).

    CAS  Article  Google Scholar 

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Acknowledgements

We are grateful for support from the National Science Foundation (NSF) Research Triangle MRSEC (DMR-1121107), Pratt–Gardner Fellowship (J.R.S.), Medtronic Inc. Fellowship in Biomedical Engineering (J.R.S.) and the NSF Graduate Research Fellowship Program (DGF1106401) (J.R.S.). A.C. acknowledges the support of the National Institutes of Health (NIH) though grants R01-GM61232, R01-EB000188 and R01-EB007205. M.R. acknowledges financial support from the NSF under grants DMR-1309892 and DMR-1436201, the NIH under grants P01-HL108808 and 1UH2HL123645, and the Cystic Fibrosis Foundation. We thank J. McDaniel, S. MacEwan and J. Genzer for their helpful discussions and for providing some of the plasmids containing genes that encode the ELPs used in this study. We also thank the Duke Light Core Microscopy Facility for fruitful discussions and help with confocal microscopy experiments.

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Contributions

J.R.S carried out experiments, protein design, expression and purification, fluorescence imaging, light scattering measurements, data analysis and manuscript preparation. N.J.C. carried out experiments, light scattering measurements, microfluidic device fabrication, data analysis, manuscript preparation and provided overall intellectual guidance. M.R. provided theoretical guidance, design of experiments, phase-diagram measurements, data interpretation and manuscript editing. A.C. provided the plasmids for protein constructs, and guidance on ELP production, ELP phase behaviour and manuscript editing. G.P.L. directed all the experiments and measurements, provided intellectual guidance, approved final edits to the manuscript and was principal investigator of the primary supporting grant. All the authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Nick J. Carroll or Gabriel P. López.

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The authors declare no competing financial interests.

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Simon, J., Carroll, N., Rubinstein, M. et al. Programming molecular self-assembly of intrinsically disordered proteins containing sequences of low complexity. Nature Chem 9, 509–515 (2017). https://doi.org/10.1038/nchem.2715

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