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Engineering protein stability with atomic precision in a monomeric miniprotein

Nature Chemical Biology volume 13, pages 764–770 (2017)Cite this article

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Abstract

Miniproteins simplify the protein-folding problem, allowing the dissection of forces that stabilize protein structures. Here we describe PPα-Tyr, a designed peptide comprising an α-helix buttressed by a polyproline II helix. PPα-Tyr is water soluble and monomeric, and it unfolds cooperatively with a midpoint unfolding temperature (TM) of 39 °C. NMR structures of PPα-Tyr reveal proline residues docked between tyrosine side chains, as designed. The stability of PPα is sensitive to modifications in the aromatic residues: replacing tyrosine with phenylalanine, i.e., changing three solvent-exposed hydroxyl groups to protons, reduces the TM to 20 °C. We attribute this result to the loss of CH–π interactions between the aromatic and proline rings, which we probe by substituting the aromatic residues with nonproteinogenic side chains. In analyses of natural protein structures, we find a preference for proline–tyrosine interactions over other proline-containing pairs, and observe abundant CH–π interactions in biologically important complexes between proline-rich ligands and SH3 and similar domains.

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Figure 1: Design of PPα combining polyproline-II and α-helices.
Figure 2: Folding and stability of PPα-Tyr and PPα variants.
Figure 3: NMR structures for the p-substituted phenylalanine variants of PPα.
Figure 4: Pairwise side chain and CH–π interactions in the Protein Data Bank.

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Acknowledgements

E.G.B. and D.N.W. are supported by a Biotechnology and Biological Sciences Research Council (BBSRC)/ERASynBio grant (BB/M005615/1); K.L.H., G.J.B., J.W.H. and D.N.W. are supported by the ERC (340764); D.N.W. is a Royal Society Wolfson Research Merit Award holder (WM140008); and K.L.P.G. is supported by the Engineering and Physical Sciences Research Council (EPSRC)-funded Bristol Chemical Synthesis Centre for Doctoral Training (EP/G036764/1). We thank BrisSynBio for access to the BBSRC/EPSRC-funded 700 MHz NMR spectrometer (BB/L01386X/1), S. Whittaker and the Henry Wellcome Building NMR Facility at the University of Birmingham for access to the Wellcome Trust–funded 900 MHz spectrometer (099185/Z/12/Z), and R. Alder and members of the Woolfson group for helpful discussions.

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Author notes

  1. Kieran L Hudson

    Present address: Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada

  2. Christopher Williams and Kieran L Hudson: These authors contributed equally to this work.

Authors and Affiliations

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

    Emily G Baker, Christopher Williams, Kieran L Hudson, Gail J Bartlett, Jack W Heal, Kathryn L Porter Goff, Matthew P Crump & Derek N Woolfson

  2. BrisSynBio, University of Bristol, Bristol, UK

    Christopher Williams, Richard B Sessions, Matthew P Crump & Derek N Woolfson

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

    Richard B Sessions & Derek N Woolfson

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Contributions

E.G.B. and D.N.W. designed the research. E.G.B. made the synthetic peptides and performed the CD spectroscopy and AUC experiments. C.W. and M.P.C. collected the NMR data. C.W., K.L.P.G. and E.G.B. analyzed the NMR data, and C.W. solved the NMR structures. K.L.H., G.J.B., D.N.W. conducted the bioinformatics. E.G.B. and R.B.S. carried out the molecular-dynamics studies. J.W.H. and E.G.B. performed the van't Hoff analyses. E.G.B. and D.N.W. wrote the paper. All authors reviewed and contributed to the manuscript.

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Correspondence to Emily G Baker or Derek N Woolfson.

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Baker, E., Williams, C., Hudson, K. et al. Engineering protein stability with atomic precision in a monomeric miniprotein. Nat Chem Biol 13, 764–770 (2017). https://doi.org/10.1038/nchembio.2380

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