Engineering an artificial, multifunctional oxidoreductase protein maquette
Redox reactions drive the energy transduction processes that are the cornerstone of biology. Oxidoreductases are of great importance not only for their biological role, but also because their charge-transferring, potential-generating functions may someday be harnessed in the inexpensive generation of clean fuels. Detailed study of natural oxidoreductases is often limited by complexity resulting from millennia of evolution. Empirical observation, however, suggests a few basic design criteria necessary to attain function. The application of these criteria was tested in the design of a minimal, hydrophilic, monomeric protein, HM-1, capable of supporting multiple redox processes, including oxygen transport, light harvesting, and electron transport, with properties predicted by the empirical models. This success demonstrates that relatively simple engineering guidelines are sufficient to design a functional protein. Sophisticated function does not come at the price of specialization. Despite its simple design, HM-1 supports diverse functions seen in natural proteins with dramatically different structures. Thus, a minimalist approach to protein design has yielded a general oxidoreductase scaffold that spontaneously folds into a helical bundle and carries out diverse biological functions without mimicking natural proteins in structure or sequence. The monomeric construction overcomes the design limitations of earlier symmetric dimer and tetramer conformations, enabling flexible modifications for varying uses. It is easily modified by changing cofactors, ligation modes, or the peptide sequence itself. The utility of an iterative, intuition-driven design process for both structural and functional improvements is demonstrated. Observed functions include covalent and non-covalent cofactor incorporation, reversible oxygen binding, and photo-induced intramolecular electron transfer. The remarkable adaptability of the scaffold suggests that nature tunes oxidoreductases principally by the adjustment of a few key parameters, knowledge of which can be applied to design synthetic and novel enzymes.