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Design and engineering of an O2 transport protein

Abstract

The principles of natural protein engineering are obscured by overlapping functions and complexity accumulated through natural selection and evolution. Completely artificial proteins offer a clean slate on which to define and test these protein engineering principles, while recreating and extending natural functions. Here we introduce this method with the design of an oxygen transport protein, akin to human neuroglobin. Beginning with a simple and unnatural helix-forming sequence with just three different amino acids, we assembled a four-helix bundle, positioned histidines to bis-histidine ligate haems, and exploited helical rotation and glutamate burial on haem binding to introduce distal histidine strain and facilitate O2 binding. For stable oxygen binding without haem oxidation, water is excluded by simple packing of the protein interior and loops that reduce helical-interface mobility. O2 affinities and exchange timescales match natural globins with distal histidines, with the remarkable exception that O2 binds tighter than CO.

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Figure 1: The design of an artificial oxygen transport protein (6).
Figure 2: Haem maquette spectra.
Figure 3: Modelling kinetics of haem ligand binding and release.

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Acknowledgements

We thank A. J. Wand for assistance in NMR measurements, P. R. Rich for FTIR measurements, M. S. Hargrove for neuroglobin reference spectra and discussions, and D. Hilvert for suggestions. This work was supported by grants from US Department of Energy, US National Institute of Health and US National Science Foundation as detailed in Supplementary Information.

Author Contributions R.L.K. and J.L.R.A. both designed proteins and performed the bulk of the measurements; K.S.R. made initial spectroscopic observations and L.A.S. contributed to spectroscopic measurements; and C.C.M. designed and performed spectroscopic measurements and analysis. Paper preparation was largely conducted by C.C.M. and P.L.D.

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Correspondence to P. Leslie Dutton.

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Koder, R., Anderson, J., Solomon, L. et al. Design and engineering of an O2 transport protein. Nature 458, 305–309 (2009). https://doi.org/10.1038/nature07841

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