Design and engineering of an O2 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. 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. In principle, the proteins that direct the heavy chemical work of the cell can be re-engineered to perform a host of other useful chemical reactions. In practice, natural proteins tend to accumulate complexity and fragility through natural selection, making it extremely difficult to re-engineer them for new purposes. Starting from a simple helix-forming sequence containing glutamate, lysine and leucine repeats, Koder et al. used de novo protein design to generate a simple unnatural oxygen transport protein, akin to human neuroglobin. The resulting four-helix bundle contains histidine residues at key positions to bind a haem cofactor, enabling the unnatural protein to bind to, and release, oxygen. The O2 on-rate, off-rate and dissociation constants are similar to those of human neuroglobin and other naturally occurring globins. The authors used de novo protein design to generate a simple unnatural oxygen transport protein, akin to human neuroglobin; the design strategy involved the assembly of a short helix-forming sequence into a four-helix bundle that contained histidine residues at key positions. The O2 on-rate, off-rate and dissociation constants are similar to those of human neuroglobin and other naturally occurring globins.