Design and engineering of an O(2) transport protein

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. We introduce this method here with the first 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 assemble a four helix bundle, position histidines to bis-his ligate hemes, and exploit helical rotation and glutamate burial on heme binding to introduce distal histidine strain and facilitate O(2) binding. For stable oxygen binding without heme oxidation, water is excluded by simple packing of the protein interior and loops that reduce helical-interface mobility. O(2) affinities and exchange timescales match natural globins with distal histidines with the remarkable exception that O(2) binds tighter than CO.