Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers


Proteins and synthetic polymers that undergo aqueous phase transitions mediate self-assembly in nature and in man-made material systems. Yet little is known about how the phase behaviour of a protein is encoded in its amino acid sequence. Here, by synthesizing intrinsically disordered, repeat proteins to test motifs that we hypothesized would encode phase behaviour, we show that the proteins can be designed to exhibit tunable lower or upper critical solution temperature (LCST and UCST, respectively) transitions in physiological solutions. We also show that mutation of key residues at the repeat level abolishes phase behaviour or encodes an orthogonal transition. Furthermore, we provide heuristics to identify, at the proteome level, proteins that might exhibit phase behaviour and to design novel protein polymers consisting of biologically active peptide repeats that exhibit LCST or UCST transitions. These findings set the foundation for the prediction and encoding of phase behaviour at the sequence level.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: The amino acid sequence of Pro- and Gly-rich proteins is characterized by discrete arrangements of Pro and Gly residues.
Figure 2: Protein polymers with repeating P–Xn–G motifs (n = 0–4) can be designed to exhibit LCST or UCST phase behaviour under physiologically relevant conditions.
Figure 3: Intrinsically disordered protein polymers can be designed to exhibit LCST or UCST phase behaviour under a wide range of conditions.
Figure 4: The phase transition behaviour of IDPPs is tunable.
Figure 5: A zwitterionic IDPP that is devoid of aromatic residues exhibits UCST phase behaviour in acidic environments.
Figure 6: IDPPs built from biologically active peptide repeats.


  1. 1

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

    CAS  Google Scholar 

  2. 2

    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  Google Scholar 

  3. 3

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

    CAS  Google Scholar 

  4. 4

    Vekilov, P. G. Phase transitions of folded proteins. Soft Matter 6, 5254–5272 (2010).

    CAS  Google Scholar 

  5. 5

    Toretsky, J. A. & Wright, P. E. Assemblages: Functional units formed by cellular phase separation. J. Cell Biol. 206, 579–588 (2014).

    CAS  Google Scholar 

  6. 6

    Frey, S., Richter, R. P. & Görlich, D. FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties. Science 314, 815–817 (2006).

    CAS  Google Scholar 

  7. 7

    Roy, D., Brooks, W. L. & Sumerlin, B. S. New directions in thermoresponsive polymers. Chem. Soc. Rev. 42, 7214–7243 (2013).

    CAS  Google Scholar 

  8. 8

    Stuart, M. A. C. et al. Emerging applications of stimuli-responsive polymer materials. Nature Mater. 9, 101–113 (2010).

    Google Scholar 

  9. 9

    McDaniel, J. R. et al. Self-assembly of thermally responsive nanoparticles of a genetically encoded peptide polymer by drug conjugation. Angew. Chem. Int. Ed. 52, 1683–1687 (2013).

    CAS  Google Scholar 

  10. 10

    MacKay, J. A. et al. Self-assembling chimeric polypeptide–doxorubicin conjugate nanoparticles that abolish tumours after a single injection. Nature Mater. 8, 993–999 (2009).

    Google Scholar 

  11. 11

    Liu, W. et al. Brachytherapy using injectable seeds that are self-assembled from genetically encoded polypeptides in situ. Cancer Res. 72, 5956–5965 (2012).

    CAS  Google Scholar 

  12. 12

    Nishida, K. et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. New Eng. J. Med. 351, 1187–1196 (2004).

    CAS  Google Scholar 

  13. 13

    Caves, J. M. et al. Elastin-like protein matrix reinforced with collagen microfibers for soft tissue repair. Biomaterials 32, 5371–5379 (2011).

    CAS  Google Scholar 

  14. 14

    Koria, P. et al. Self-assembling elastin-like peptides growth factor chimeric nanoparticles for the treatment of chronic wounds. Proc. Natl Acad. Sci. USA 108, 1034–1039 (2011).

    CAS  Google Scholar 

  15. 15

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

    CAS  Google Scholar 

  16. 16

    Bellucci, J. J., Amiram, M., Bhattacharyya, J., McCafferty, D. & Chilkoti, A. Three-in-one chromatography-free purification, tag removal, and site-specific modification of recombinant fusion proteins using sortase A and elastin-like polypeptides. Angew. Chem. Int. Ed. 52, 3703–3708 (2013).

    CAS  Google Scholar 

  17. 17

    Stayton, P. S. et al. Control of protein–ligand recognition using a stimuli-responsive polymer. Nature 378, 472–474 (1995).

    CAS  Google Scholar 

  18. 18

    Dill, K. A. & MacCallum, J. L. The protein-folding problem, 50 years on. Science 338, 1042–1046 (2012).

    CAS  Google Scholar 

  19. 19

    Habchi, J., Tompa, P., Longhi, S. & Uversky, V. N. Introducing protein intrinsic disorder. Chem. Rev. 114, 6561–6588 (2014).

    CAS  Google Scholar 

  20. 20

    Li, N. K., Quiroz, F. G., Hall, C. K., Chilkoti, A. & Yingling, Y. G. Molecular description of the LCST behaviour of an elastin-like polypeptide. Biomacromolecules 15, 3522–3530 (2014).

    CAS  Google Scholar 

  21. 21

    Muiznieks, L. D. & Keeley, F. W. Proline periodicity modulates the self-assembly properties of elastin-like polypeptides. J. Biol. Chem. 285, 39779–39789 (2010).

    CAS  Google Scholar 

  22. 22

    Dutta, N. K. et al. A genetically engineered protein responsive to multiple stimuli. Angew. Chem. Int. Ed. 50, 4428–4431 (2011).

    CAS  Google Scholar 

  23. 23

    Rauscher, S., Baud, S., Miao, M., Keeley, F. W. & Pomès, R. Proline and glycine control protein self-organization into elastomeric or amyloid fibrils. Structure 14, 1667–1676 (2006).

    CAS  Google Scholar 

  24. 24

    De Las Heras Alarcon, C., Pennadam, S. & Alexander, C. Stimuli responsive polymers for biomedical applications. Chem. Soc. Rev. 34, 276–285 (2005).

    Google Scholar 

  25. 25

    Amiram, M., Quiroz, F. G., Callahan, D. J. & Chilkoti, A. A highly parallel method for synthesizing DNA repeats enables the discovery of ‘smart’ protein polymers. Nature Mater. 10, 141–148 (2011).

    CAS  Google Scholar 

  26. 26

    Balu, R. et al. An16-resilin: An advanced multi-stimuli-responsive resilin-mimetic protein polymer. Acta Biomater. 10, 4768–4777 (2014).

    CAS  Google Scholar 

  27. 27

    Urry, D. W. et al. Hydrophobicity scale for proteins based on inverse temperature transitions. Biopolymers 32, 1243–1250 (1992).

    CAS  Google Scholar 

  28. 28

    Das, R. K. & Pappu, R. V. Conformations of intrinsically disordered proteins are influenced by linear sequence distributions of oppositely charged residues. Proc. Natl Acad. Sci. USA 110, 13392–13397 (2013).

    CAS  Google Scholar 

  29. 29

    Yokoi, H., Kinoshita, T. & Zhang, S. Dynamic reassembly of peptide RADA16 nanofiber scaffold. Proc. Natl Acad. Sci. USA 102, 8414–8419 (2005).

    CAS  Google Scholar 

  30. 30

    Müller-Späth, S. et al. Charge interactions can dominate the dimensions of intrinsically disordered proteins. Proc. Natl Acad. Sci. USA 107, 14609–14614 (2010).

    Google Scholar 

  31. 31

    Möglich, A., Joder, K. & Kiefhaber, T. End-to-end distance distributions and intrachain diffusion constants in unfolded polypeptide chains indicate intramolecular hydrogen bond formation. Proc. Natl Acad. Sci. USA 103, 12394–12399 (2006).

    Google Scholar 

  32. 32

    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  Google Scholar 

  33. 33

    Seuring, J. & Agarwal, S. First example of a universal and cost-effective approach: Polymers with tunable upper critical solution temperature in water and electrolyte solution. Macromolecules 45, 3910–3918 (2012).

    CAS  Google Scholar 

  34. 34

    Shimada, N. et al. Ureido-derivatized polymers based on both poly (allylurea) and poly (L-citrulline) exhibit UCST-type phase transition behaviour under physiologically relevant conditions. Biomacromolecules 12, 3418–3422 (2011).

    CAS  Google Scholar 

  35. 35

    Seuring, J. & Agarwal, S. Polymers with upper critical solution temperature in aqueous solution: Unexpected properties from known building blocks. ACS Macro Lett. 2, 597–600 (2013).

    CAS  Google Scholar 

  36. 36

    Seuring, J. & Agarwal, S. Polymers with upper critical solution temperature in aqueous solution. Macromol. Rapid Commun. 33, 1898–1920 (2012).

    CAS  Google Scholar 

  37. 37

    Azzaroni, O., Brown, A. A. & Huck, W. T. UCST wetting transitions of polyzwitterionic brushes driven by self-association. Angew. Chem. 118, 1802–1806 (2006).

    Google Scholar 

  38. 38

    Lowe, A. B. & McCormick, C. L. Synthesis and solution properties of zwitterionic polymers. Chem. Rev. 102, 4177–4190 (2002).

    CAS  Google Scholar 

  39. 39

    Mason, P., Neilson, G., Dempsey, C., Barnes, A. & Cruickshank, J. The hydration structure of guanidinium and thiocyanate ions: Implications for protein stability in aqueous solution. Proc. Natl Acad. Sci. USA 100, 4557–4561 (2003).

    CAS  Google Scholar 

  40. 40

    Mahadevi, A. S. & Sastry, G. N. Cation-π interaction: Its role and relevance in chemistry, biology, and material science. Chem. Rev. 113, 2100–2138 (2013).

    CAS  Google Scholar 

  41. 41

    Kao, W. J., Lee, D., Schense, J. C. & Hubbell, J. A. Fibronectin modulates macrophage adhesion and FBGC formation: The role of RGD, PHSRN, and PRRARV domains. J. Biomed. Mater. Res. 55, 79–88 (2001).

    CAS  Google Scholar 

  42. 42

    Iwamoto, Y. et al. YIGSR, a synthetic laminin pentapeptide, inhibits experimental metastasis formation. Science 238, 1132–1134 (1987).

    CAS  Google Scholar 

  43. 43

    Pierschbacher, M. D. & Ruoslahti, E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 309, 30–33 (1983).

    Google Scholar 

  44. 44

    Lee, B. W. et al. Strongly binding cell-adhesive polypeptides of programmable valencies. Angew. Chem. Int. Ed. 49, 1971–1975 (2010).

    CAS  Google Scholar 

  45. 45

    van der Lee, R. et al. Classification of intrinsically disordered regions and proteins. Chem. Rev. 114, 6589–6631 (2014).

    CAS  Google Scholar 

  46. 46

    Brassart, B. et al. Conformational dependence of collagenase (matrix metalloproteinase-1) up-regulation by elastin peptides in cultured fibroblasts. J. Biol. Chem. 276, 5222–5227 (2001).

    CAS  Google Scholar 

  47. 47

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

    CAS  Google Scholar 

  48. 48

    Callahan, D. J. et al. Triple stimulus-responsive polypeptide nanoparticles that enhance intratumoral spatial distribution. Nano Lett. 12, 2165–2170 (2012).

    CAS  Google Scholar 

  49. 49

    Crick, S. L., Ruff, K. M., Garai, K., Frieden, C. & Pappu, R. V. Unmasking the roles of N-and C-terminal flanking sequences from exon 1 of huntingtin as modulators of polyglutamine aggregation. Proc. Natl Acad. Sci. USA 110, 20075–20080 (2013).

    CAS  Google Scholar 

  50. 50

    Bates, F. S. et al. Multiblock polymers: Panacea or Pandora’s box? Science 336, 434–440 (2012).

    CAS  Google Scholar 

  51. 51

    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  Google Scholar 

  52. 52

    Tong, R., Chiang, H. H. & Kohane, D. S. Photoswitchable nanoparticles for in vivo cancer chemotherapy. Proc. Natl Acad. Sci. USA 110, 19048–19053 (2013).

    CAS  Google Scholar 

  53. 53

    Wong, C. et al. Multistage nanoparticle delivery system for deep penetration into tumor tissue. Proc. Natl Acad. Sci. USA 108, 2426–2431 (2011).

    CAS  Google Scholar 

  54. 54

    Clark, J. I. Self-assembly of protein aggregates in ageing disorders: The lens and cataract model. Phil. Trans. R. Soc. B 368, 20120104 (2013).

    Google Scholar 

  55. 55

    Smith, F. J. et al. Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris. Nature Genet. 38, 337–342 (2006).

    CAS  Google Scholar 

  56. 56

    Kyte, J. & Doolittle, R. F. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132 (1982).

    CAS  Google Scholar 

  57. 57

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

    CAS  Google Scholar 

Download references


F.G.Q. thanks K. Zhu for his assistance with the preparation of Fig. 4d. This work was funded by the NIH through grant # GM061232 to A.C. and by the NSF through the Research Triangle MRSEC (NSF DMR-11-21107).

Author information




F.G.Q. designed and performed experiments, analysed data and wrote the manuscript. A.C. analysed data and wrote the manuscript.

Corresponding author

Correspondence to Ashutosh Chilkoti.

Ethics declarations

Competing interests

The authors hold US patent no. 8,470,967 that covers many of the peptide sequences described in this Article.

Supplementary information

Supplementary Information

Supplementary Information (PDF 2888 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Quiroz, F., Chilkoti, A. Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers. Nature Mater 14, 1164–1171 (2015).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing