The dispersion of the water 1H longitudinal relaxation rate in the frequency range 2100 MHz has been measured in aqueous solutions of bovine pancreatic trypsin inhibitor (BPTI) and a mutant protein (G36S) lacking one of the four internal water molecules. The 1H relaxation dispersion has also been measured for BPTI in a series of H2O/D2O mixtures. The quantitative analysis of these data resolve the major controversies in the interpretation of water 1H relaxation data from protein solutions and has implications for medical magnetic resonance imaging. Three principal conclusions are drawn. First, as previously found for the water 2H and 17O dispersions, the BPTI G36S difference 1H dispersion can be quantitatively accounted for by a single, fully ordered, internal water molecule (W122). The intrinsic relaxation rate of these water protons is ca. 70% intramolecular, with the intramolecular dipole coupling constant as in ice, and ca. 30% intermolecular, with significant dipole couplings to many BPTI protons. Second, exchanging protons in the protein make a substantial contribution to the observed water 1H relaxation rate. This contribution should be dominant even at neutral pH for most proteins. Third, the effect of intermolecular dipole couplings with protein protons is additive, and cross-relaxation effects are negligible. A theoretical analysis of dipole relaxation in a multispin system undergoing chemical exchange with an abundant bulk phase shows that this conclusion holds generally within the regime of motional narrowing theory.
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