Skip to main content

Thank you for visiting nature.com. 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.

De novo design of discrete, stable 310-helix peptide assemblies

Abstract

The α-helix is pre-eminent in structural biology1 and widely exploited in protein folding2, design3 and engineering4. Although other helical peptide conformations do exist near to the α-helical region of conformational space—namely, 310-helices and π-helices5—these occur much less frequently in protein structures. Less favourable internal energies and reduced tendencies to pack into higher-order structures mean that 310-helices rarely exceed six residues in length in natural proteins, and that they tend not to form normal supersecondary, tertiary or quaternary interactions. Here we show that despite their absence in nature, synthetic peptide assemblies can be built from 310-helices. We report the rational design, solution-phase characterization and an X-ray crystal structure for water-soluble bundles of 310-helices with consolidated hydrophobic cores. The design uses six-residue repeats informed by analysing 310-helical conformations in known protein structures, and incorporates α-aminoisobutyric acid residues. Design iterations reveal a tipping point between α-helical and 310-helical folding, and identify features required for stabilizing assemblies of 310-helices. This work provides principles and rules to open opportunities for designing into this hitherto unexplored region of protein-structure space.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Analysis of the PDB and design principles for 310-helices.
Fig. 2: Biophysical characterization of de-novo-designed peptides.
Fig. 3: Crystal structures of de-novo-designed peptides.

Data availability

The coordinate and structure factor files are available from the Research Collaboratory for Structural Bioinformatics PDB under the following accession codes: CCTri-TypeN-LaLd (PDB ID: 7QDK); D-310HD (PDB ID: 7QDI); PK-10 + PK-11 (PDB ID: 7QDJ). The list of PDB files for the bioinformatic analyses was downloaded from the Pisces server (http://dunbrack.fccc.edu/pisces/download/). Source data are provided with this paper. Additional data to generate figures in the Supplementary Information are available at http://coiledcoils.chm.bris.ac.uk/SI-data/PK-310/.

Code availability

The customized scripts used for bioinformatic analyses are available at http://coiledcoils.chm.bris.ac.uk/SI-data/PK-310/.

References

  1. Pauling, L., Corey, R. B. & Branson, H. R. The structure of proteins; two hydrogen-bonded helical configurations of the polypeptide chain. Proc. Natl Acad. Sci. USA 37, 205–211 (1951).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. Chakrabartty, A. & Baldwin, R. L. Stability of alpha-helices. Adv. Protein Chem. 46, 141–176 (1995).

    CAS  PubMed  Article  Google Scholar 

  3. Korendovych, I. V. & DeGrado, W. F. De novo protein design, a retrospective. Q. Rev. Biophys. 53, e3 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. Lapenta, F., Aupic, J., Strmsek, Z. & Jerala, R. Coiled coil protein origami: from modular design principles towards biotechnological applications. Chem. Soc. Rev. 47, 3530–3542 (2018).

    CAS  PubMed  Article  Google Scholar 

  5. Schulz, G. E. & Schirmer, R. H. Principles of Protein Structure (Springer, 1979).

  6. Scholtz, J. M. & Baldwin, R. L. The mechanism of alpha-helix formation by peptides. Annu. Rev. Biophys. Biomol. Struct. 21, 95–118 (1992).

    CAS  PubMed  Article  Google Scholar 

  7. Woolfson, D. N. A brief history of de novo protein design: minimal, rational, and computational. J. Mol. Biol. 433, 167160 (2021).

    CAS  PubMed  Article  Google Scholar 

  8. Dawson, W. M., Rhys, G. G. & Woolfson, D. N. Towards functional de novo designed proteins. Curr. Opin. Chem. Biol. 52, 102–111 (2019).

    CAS  PubMed  Article  Google Scholar 

  9. Ramachandran, G. N., Ramakrishnan, C. & Sasisekharan, V. Stereochemistry of polypeptide chain configurations. J. Mol. Biol. 7, 95–99 (1963).

    CAS  PubMed  Article  Google Scholar 

  10. Kuster, D. J., Liu, C., Fang, Z., Ponder, J. W. & Marshall, G. R. High-resolution crystal structures of protein helices reconciled with three-centered hydrogen bonds and multipole electrostatics. PLoS ONE 10, e0123146 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  11. Burley, S. K. et al. RCSB Protein Data Bank: powerful new tools for exploring 3D structures of biological macromolecules for basic and applied research and education in fundamental biology, biomedicine, biotechnology, bioengineering and energy sciences. Nucleic Acids Res. 49, D437–D451 (2021).

    CAS  PubMed  Article  Google Scholar 

  12. Chothia, C., Levitt, M. & Richardson, D. Helix to helix packing in proteins. J. Mol. Biol. 145, 215–250 (1981).

    CAS  PubMed  Article  Google Scholar 

  13. Orengo, C. A. et al. CATH–a hierarchic classification of protein domain structures. Structure 5, 1093–1108 (1997).

    CAS  PubMed  Article  Google Scholar 

  14. Gessmann, R., Axford, D., Owen, R. L., Bruckner, H. & Petratos, K. Four complete turns of a curved 310-helix at atomic resolution: the crystal structure of the peptaibol trichovirin I-4A in a polar environment suggests a transition to α-helix for membrane function. Acta Crystallogr. D 68, 109–116 (2012).

    CAS  PubMed  Article  Google Scholar 

  15. Toniolo, C. & Brückner, H. Peptaibiotics (Wiley, 2009).

  16. Toniolo, C. & Benedetti, E. The polypeptide 310-helix. Trends Biochem. Sci. 16, 350–353 (1991).

    CAS  PubMed  Article  Google Scholar 

  17. Gessmann, R., Bruckner, H. & Petratos, K. The crystal structure of Z-(Aib)10-OH at 0.65 Å resolution: three complete turns of 310-helix. J. Pept. Sci. 22, 76–81 (2016).

    CAS  PubMed  Article  Google Scholar 

  18. Solà, J., Helliwell, M. & Clayden, J. Interruption of a 310-helix by a single Gly residue in a poly-Aib motif: a crystallographic study. Biopolymers 95, 62–69 (2011).

    PubMed  Article  CAS  Google Scholar 

  19. Pike, S. J., Boddaert, T., Raftery, J., Webb, S. J. & Clayden, J. Participation of non-aminoisobutyric acid (Aib) residues in the 310 helical conformation of Aib-rich foldamers: a solid state study. New J. Chem. 39, 3288–3294 (2015).

    CAS  Article  Google Scholar 

  20. Karle, I. L., Flippen-Anderson, J. L., Gurunath, R. & Balaram, P. Facile transition between 310- and α-helix: structures of 8-, 9-, and 10-residue peptides containing the -(Leu-Aib-Ala)2-Phe-Aib-fragment. Protein Sci. 3, 1547–1555 (1994).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. Toniolo, C. et al. Preferred conformation of the terminally blocked (Aib)10 homo-oligopeptide: a long, regular 310-helix. Biopolymers 31, 129–138 (1991).

    CAS  Article  Google Scholar 

  22. Nagaraj, R. & Balaram, P. Alamethicin, a transmembrane channel. Acc. Chem. Res. 14, 356–362 (1981).

    CAS  Article  Google Scholar 

  23. Toniolo, C. et al. Conformation of pleionomers of .alpha.-aminoisobutyric acid. Macromolecules 18, 895–902 (1985).

    ADS  CAS  Article  Google Scholar 

  24. Karle, I. L. & Balaram, P. Structural characteristics of alpha-helical peptide molecules containing Aib residues. Biochemistry 29, 6747–6756 (1990).

    CAS  PubMed  Article  Google Scholar 

  25. Byrne, L. et al. Foldamer-mediated remote stereocontrol: >1,60 asymmetric induction. Angew. Chem. Int. Ed. 53, 151–155 (2014).

    CAS  Article  Google Scholar 

  26. Lister, F. G. A., Le Bailly, B. A. F., Webb, S. J. & Clayden, J. Ligand-modulated conformational switching in a fully synthetic membrane-bound receptor. Nat. Chem. 9, 420–425 (2017).

    CAS  Article  Google Scholar 

  27. De Poli, M. et al. Conformational photoswitching of a synthetic peptide foldamer bound within a phospholipid bilayer. Science 352, 575–580 (2016).

    ADS  PubMed  Article  CAS  Google Scholar 

  28. Formaggio, F. et al. The first water-soluble 310-helical peptides. Chemistry 6, 4498–4504 (2000).

    CAS  PubMed  Article  Google Scholar 

  29. Zieleniewski, F., Woolfson, D. N. & Clayden, J. Automated solid-phase concatenation of Aib residues to form long, water-soluble, helical peptides. Chem. Commun. 56, 12049–12052 (2020).

    CAS  Article  Google Scholar 

  30. Woolfson, D. N. Coiled-coil design: updated and upgraded. Subcell. Biochem. 82, 35–61 (2017).

    CAS  PubMed  Article  Google Scholar 

  31. Fletcher, J. M. et al. A basis set of de novo coiled-coil peptide oligomers for rational protein design and synthetic biology. ACS Synth. Biol. 1, 240–250 (2012).

    CAS  PubMed  Article  Google Scholar 

  32. Harbury, P. B., Zhang, T., Kim, P. S. & Alber, T. A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. Science 262, 1401–1407 (1993).

    ADS  CAS  PubMed  Article  Google Scholar 

  33. Kumar, P. & Woolfson, D. N. Socket2: a program for locating, visualising, and analysing coiled-coil interfaces in protein structures. Bioinformatics 37, 4575–4577 (2021).

    CAS  PubMed Central  Article  Google Scholar 

  34. Swanson, C. J. & Sivaramakrishnan, S. Harnessing the unique structural properties of isolated alpha-helices. J. Biol. Chem. 289, 25460–25467 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. Brown, R. A., Marcelli, T., De Poli, M., Sola, J. & Clayden, J. Induction of unexpected left-handed helicity by an N-terminal L-amino acid in an otherwise achiral peptide chain. Angew. Chem. Int. Ed. 51, 1395–1399 (2012).

    CAS  Article  Google Scholar 

  36. Thomson, A. R. et al. Computational design of water-soluble alpha-helical barrels. Science 346, 485–488 (2014).

    ADS  CAS  PubMed  Article  Google Scholar 

  37. Thomas, F. et al. De novo-designed alpha-helical barrels as receptors for small molecules. ACS Synth. Biol. 7, 1808–1816 (2018).

    CAS  PubMed  Article  Google Scholar 

  38. Enkhbayar, P., Hikichi, K., Osaki, M., Kretsinger, R. H. & Matsushima, N. 310-helices in proteins are parahelices. Proteins 64, 691–699 (2006).

    CAS  PubMed  Article  Google Scholar 

  39. Kumar, P. & Bansal, M. HELANAL-Plus: a web server for analysis of helix geometry in protein structures. J. Biomol. Struct. Dyn. 30, 773–783 (2012).

    CAS  PubMed  Article  Google Scholar 

  40. Lupas, A. N. & Gruber, M. The structure of alpha-helical coiled coils. Adv. Protein Chem. 70, 37–78 (2005).

    CAS  PubMed  Article  Google Scholar 

  41. Hunter, C. A. & Sanders, J. K. M. The nature of π-π interactions. J. Am. Chem. Soc. 112, 5525–5534 (1990).

    CAS  Article  Google Scholar 

  42. Mortenson, D. E. et al. High-resolution structures of a heterochiral coiled coil. Proc. Natl Acad. Sci. USA 112, 13144–13149 (2015).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. Fox, R. O. Jr. & Richards, F. M. A voltage-gated ion channel model inferred from the crystal structure of alamethicin at 1.5-Å resolution. Nature 300, 325–330 (1982).

    ADS  CAS  PubMed  Article  Google Scholar 

  44. Bunkoczi, G., Schiell, M., Vertesy, L. & Sheldrick, G. M. Crystal structures of cephaibols. J. Pept. Sci. 9, 745–752 (2003).

    CAS  PubMed  Article  Google Scholar 

  45. Mendel, D., Ellman, J. & Schultz, P. G. Protein biosynthesis with conformationally restricted amino acids. J. Am. Chem. Soc. 115, 4359–4360 (1993).

    CAS  Article  Google Scholar 

  46. Leonard, D. J., Ward, J. W. & Clayden, J. Asymmetric α-arylation of amino acids. Nature 562, 105–109 (2018).

    ADS  CAS  PubMed  Article  Google Scholar 

  47. Collie, G. W. et al. Shaping quaternary assemblies of water-soluble non-peptide helical foldamers by sequence manipulation. Nat. Chem. 7, 871–878 (2015).

    CAS  PubMed  Article  Google Scholar 

  48. Wang, P. S. & Schepartz, A. β-Peptide bundles: Design. Build. Analyze. Biosynthesize. Chem. Commun. 52, 7420–7432 (2016).

    CAS  Article  Google Scholar 

  49. Chandramouli, N. et al. Iterative design of a helically folded aromatic oligoamide sequence for the selective encapsulation of fructose. Nat. Chem. 7, 334–341 (2015).

    CAS  PubMed  Article  Google Scholar 

  50. Girvin, Z. C., Andrews, M. K., Liu, X. & Gellman, S. H. Foldamer-templated catalysis of macrocycle formation. Science 366, 1528–1531 (2019).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. Wang, G. & Dunbrack, R. L. Jr. PISCES: a protein sequence culling server. Bioinformatics 19, 1589–1591 (2003).

    CAS  PubMed  Article  Google Scholar 

  52. Joosten, R. P. et al. A series of PDB related databases for everyday needs. Nucleic Acids Res. 39, D411–419 (2011).

    CAS  PubMed  Article  Google Scholar 

  53. Hunter, J. D. Matplotlib: a 2D graphics environment. Comput. Sci. Eng. 9, 90–95 (2007).

    Article  Google Scholar 

  54. The PyMOL Molecular Graphics System Open-Source v2.4.0 (Schrödinger, 2021).

  55. Schuck, P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys. J. 78, 1606–1619 (2000).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  56. Laue, T., Shah, B., Ridgeway, T. & Pelletier, S. in Analytical Ultracentrifugation in Biochemistry and Polymer Science (eds Harding, S. E. et al.) 90–125 (Royal Society of Chemistry, 1992).

  57. Zhao, H., Piszczek, G. & Schuck, P. SEDPHAT–a platform for global ITC analysis and global multi-method analysis of molecular interactions. Methods 76, 137–148 (2015).

    CAS  PubMed  Article  Google Scholar 

  58. Winter, G. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, 186–190 (2010).

    CAS  Article  Google Scholar 

  59. Battye, T. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D 67, 271–281 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  60. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006).

    PubMed  Article  CAS  Google Scholar 

  61. Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D 69, 1204–1214 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  62. Evans, P. R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D 67, 282–292 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  63. Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  64. Sheldrick, G. M. SHELXT - integrated space-group and crystal-structure determination. Acta Crystallogr. A 71, 3–8 (2015).

    MATH  Article  CAS  Google Scholar 

  65. Dolomanov, O. V., Bourhis, L. J., Gildea, R. J., Howard, J. A. K. & Puschmann, H. OLEX2: a complete structure solution, refinement and analysis program. J. Appl. Crystallogr. 42, 339–341 (2009).

    CAS  Article  Google Scholar 

  66. Sammito, M. et al. ARCIMBOLDO_LITE: single-workstation implementation and use. Acta Crystallogr. D 71, 1921–1930 (2015).

    CAS  PubMed  Article  Google Scholar 

  67. Caballero, I. et al. ARCIMBOLDO on coiled coils. Acta Crystallogr. D 74, 194–204 (2018).

    CAS  Article  Google Scholar 

  68. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  69. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  70. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  71. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D 75, 861–877 (2019).

    CAS  Article  Google Scholar 

  72. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  73. Joosten, R. P., Long, F., Murshudov, G. N. & Perrakis, A. The PDB_REDO server for macromolecular structure model optimization. IUCrJ 1, 213–220 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

P.K. and D.N.W. are supported by Biotechnology and Biological Sciences Research Council (BB/R00661X/1) and European Research Council (340764) grants to D.N.W. D.N.W. is also supported by BrisSynBio, a Biotechnology and Biological Sciences Research Council/Engineering and Physical Sciences Research Council (EPSRC)-financed Synthetic Biology Research Centre (BB/L01386X/1), and a Royal Society Wolfson Research Merit Award (WM140008). J.C. is supported by the European Research Council Advanced Grant DOGMATRON (agreement no. 884786) and an EPSRC Programme Grant (EP/P027067/1). We thank the University of Bristol School of Chemistry Mass Spectrometry Facility for access to the EPSRC-financed Bruker Ultraflex MALDI-TOF/TOF instrument (EP/K03927X/1), and BrisSynBio for access to peptide synthesizers. We thank C. Williams for collecting one-dimensional 1H nuclear magnetic resonance spectra. We thank Diamond Light Source for access to beamlines I03, I04, I04-1 and I24 (Proposal 23269) and M. Warren from I19 who helped N.G.P. with the direct methods solution. We thank T. Yeates (University of California, Los Angeles), K. Gupta, C. Tölzer, F. Zieleniewski and members of the Clayden and Woolfson laboratories and BrisSynBio for helpful discussions.

Author information

Authors and Affiliations

Authors

Contributions

P.K., J.C. and D.N.W. conceived the project. P.K. and D.N.W. designed the bioinformatics analyses, which were performed by P.K. P.K. and D.N.W. designed the sequences, which were synthesized, characterized and crystallized by P.K. P.K. and N.G.P. solved the X-ray crystal structures. P.K., J.C. and D.N.W. wrote the manuscript, which was read by all authors.

Corresponding authors

Correspondence to Prasun Kumar or Derek N. Woolfson.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks Giovanna Ghirlanda and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

This file contains the following seven sections: Section 1, Bioinformatics analyses; Section 2, Analytical high-pressure liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization - time of flight (MALDI-TOF); Section 3, Circular dichroism (CD) spectroscopy; Section 4, Analytical ultracentrifugation (AUC); Section 5, DPH-binding analyses; Section 6, Structural analyses of 310-helix bundle; Section 7, Tables.

Reporting Summary

Peer Review File

Supplementary Code

This file contains customized scripts used for bioinformatic analyses.

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kumar, P., Paterson, N.G., Clayden, J. et al. De novo design of discrete, stable 310-helix peptide assemblies. Nature (2022). https://doi.org/10.1038/s41586-022-04868-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41586-022-04868-x

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

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