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

The problem of the interaction of water solvent with proteins has been addressed by investigating the water H-1 nuclear magnetic relaxation dispersion (NMRD) profiles of cytochrome c solutions. It is shown that the H-1 NMRD profiles are accounted for by 1, a sizeable contribution from exchangeable protein protons (mostly from lysine side chains) and 2, a modest contribution from long-lived water. It is also shown that the number of exchangeable protons 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 concentration. This additional contribution arises mostly from backbone protons, as evidenced by high resolution NMR data which provide significant and independent data on the structure and the dynamic behaviour of the partly unfolded oxidized protein. Higher accessibility to short lived water molecules is proposed also. For the analysis of the H-1 NMRD data a complete relaxation matrix approach is presented that is analogous, but not identical, to one 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.