Comparative laboratory evolution of ordered and disordered enzymes.

Intrinsically disordered proteins are ubiquitous in nature. To assess potential evolutionary advantages and disadvantages of structural disorder under controlled laboratory conditions, we directly compared the evolvability of weakly active ordered and disordered variants of dihydrofolate reductase by genetic selection. The circularly permuted Escherichia coli enzyme, which exists as a molten globule in the absence of ligands, and a well folded deletion mutant of the Bacillus stearothermophilus enzyme served as starting points. Both scaffolds evolved at similar rates and to similar extents, reaching near-native activity after three rounds of mutagenesis and selection. Surprisingly, however, the starting structural properties of the two scaffolds changed only marginally during optimization. Although the ordered and disordered proteins accumulated distinct sets of mutations, the changes introduced likely improved catalytic efficiency indirectly in both cases by bolstering the network of dynamic conformational fluctuations that productively couple into the reaction coordinate.