Installing hydrolytic activity into a completely de novo protein framework

Abstract

The design of enzyme-like catalysts tests our understanding of sequence-to-structure/function relationships in proteins. Here we install hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprises seven helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine–histidine–glutamic acid triads on each helix, hydrolyses p-nitrophenyl acetate with catalytic efficiencies that match the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis.

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Figure 1: Solution-phase biophysical characterization of the designed peptides.
Figure 2: X-ray crystal structures of CC-Hept mutants.
Figure 3: Mechanistic and kinetic analysis of the reaction of the designed peptides with pNPA.
Figure 4: Characterization and hydrolytic evaluation of CC-Hept–hCys–His–Glu and CC-Hept–βMCys–His–Glu.

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Acknowledgements

A.J.B. thanks the Bristol Chemical Synthesis Centre for Doctoral Training funded by the Engineering and Physical Sciences Research Council (EP/G036764/1) and the University of Bristol for the provision of a PhD studentship. A.J.B., A.R.T., W.M.D. and D.N.W. are supported by the European Research Council (340764). D.N.W. holds a Royal Society Wolfson Research Merit Award. We thank the Diamond Light Source for access to beamlines I03, I04 and I24 (award MX-8922), and F. Thomas and members of the Woolfson group for helpful discussions.

Author information

A.J.B., A.R.T. and D.N.W. designed the research. A.J.B. synthesized the peptides and the βMCys amino acid and performed the solution-phase biophysical analyses. A.J.B. and W.M.D. performed the kinetic experiments. A.J.B. and R.L.B. solved the X-ray crystal structures. A.J.B. and D.N.W. wrote the manuscript. All the authors analysed the data, and reviewed and contributed to the manuscript.

Correspondence to Derek N. Woolfson.

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Burton, A., Thomson, A., Dawson, W. et al. Installing hydrolytic activity into a completely de novo protein framework. Nature Chem 8, 837–844 (2016) doi:10.1038/nchem.2555

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