Determination of Protein Surface Hydration by Systematic Charge Mutations

Protein surface hydration is critical to its structural stability, flexibility, dynamics, and function. Recent observations of surface solvation on picosecond time scales have evoked debate on the origin of such relatively slow motions, from hydration water or protein charged side chains, especially with molecular dynamics simulations. Here we used a unique nuclease with a single tryptophan as a local probe and systematically mutated three neighboring charged residues to differentiate the contributions from hydration water and charged side chains. By various mutations of one, two, and all three charged residues, we observed slight increases in the total tryptophan Stokes shifts with fewer neighboring charged residue(s) and found insensitivity of charged side chains to the relaxation patterns. The dynamics is correlated with hydration water relaxation with the slowest time in a dense charged environment and the fastest time at a hydrophobic site. On such picosecond time scales, the protein surface motion is restricted. The total Stokes shifts are dominantly from hydration water relaxation and the slow dynamics is from water-driven relaxation, coupled to local protein fluctuations.