Water-protein interaction in native and partially unfolded equine cytochrome c

The problem of the interaction of water solvent with proteins has been addr essed by investigating the water H-1 nuclear magnetic relaxation dispersion (NMRD) profiles of cytochrome c solutions. It is shown that the H-1 NMRD p rofiles are accounted for by 1, a sizeable contribution from exchangeable p rotein protons (mostly from lysine side chains) and 2, a modest contributio n from long-lived water. It is also shown that the number of exchangeable p rotons is sizeably increased in the oxidized but not in the reduced protein in the presence of the unfolding agent guanidinium chloride at a 3 M conce ntration. This additional contribution arises mostly from backbone protons, as evidenced by high resolution NMR data which provide significant and ind ependent data on the structure and the dynamic behaviour of the partly unfo lded oxidized protein. Higher accessibility to short lived water molecules is proposed also. For the analysis of the H-1 NMRD data a complete relaxati on matrix approach is presented that is analogous, but not identical, to on e recently described. This approach permits the simultaneous incorporation of exchangeable protein protons and an unlimited number of water molecules in pre-defined protein binding sites.