A cross-section of the fitness landscape of dihydrofolate reductase

In vitro molecular evolution is regarded as a hill-climbing on a fitness la ndscape in sequence space, where the "fitness" is a quantitative measure of a certain physicochemical property of a biopolymer. We analyzed a "cross-s ection" of the enzymatic activity landscape of dihydrofolate reductase (DHF R) by using a method of analysis of a fitness landscape. We limited the seq uence space of interest to the five-dimensional sequence space, where the c oordinate corresponds to the 1st, 16th, 20th, 42nd and 92nd site in the DHF R sequence. Thirty six mutants mapped into the limited sequence space were taken in the analysis. As a result, the cross-section is of the rough Mt Fu ji type based on the mutational additivity. The ratio of the mean slope to the roughness is 2.8 and the Z-score of the original ratio against a distri bution of random references is 7.0, which indicates a large statistical sig nificance. The existence of such a cross-section was discussed in terms of the occurrence probability of sets of five sites distantly separated from e ach other on the DHFR 3D structure. Our results support the effectiveness o f the evolution strategy which exploits the accumulation of advantageous si ngle point mutations in such a cross-section.