Abstract
Enzymatic activity requires a precise balance between flexibility and stability is a widely accepted concept, however key question remain: How do motions on different timescales relate to each other and contribute to this balance? Have proteins evolved so that substates necessary for activity are preferable accessible? Here we report experimental evidence and corresponding computational predictions for multiple substates of an enzyme along the reaction trajectory. Strikingly we find that the enzyme indeed does not randomly sample conformational space but that there are preferred motions along the reaction trajectory. The timescale and amplitude of motion were characterized by a combination of NMR relaxation, single molecule FRET experiments, crystallography and MD simulations. The determined predisposition of enzymes to move in the direction utilized for catalysis may be a key factor for the efficiency of biocatalysts.
Characterization of backbone order parameters revealed several highly flexible spots. Many of them are located in the exact regions where local conformational change is required for the larger changes that happen in the microsecond time regime. These results suggest that local ps-ns fluctuations are the origin of the slower, larger amplitude domain motions.