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

Nature volume 607, pages 387–392 (2022)Cite this article

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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.

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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.

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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/.

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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

  1. School of Chemistry, University of Bristol, Bristol, UK

    Prasun Kumar, Jonathan Clayden & Derek N. Woolfson

  2. Diamond Light Source, Didcot, UK

    Neil G. Paterson

  3. School of Biochemistry, University of Bristol, Bristol, UK

    Derek N. Woolfson

  4. BrisSynBio, University of Bristol, Bristol, UK

    Derek N. Woolfson

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  1. Prasun Kumar
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  4. Derek N. Woolfson
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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.

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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.

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Kumar, P., Paterson, N.G., Clayden, J. et al. De novo design of discrete, stable 310-helix peptide assemblies. Nature 607, 387–392 (2022). https://doi.org/10.1038/s41586-022-04868-x

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