Buried water molecules constitute a highly conserved, integral part of nearly all known protein structures. Such water molecules exchange with external solvent as a result of protein conformational fluctuations. We report here the results of water (17)O and (2)H magnetic relaxation dispersion measurements on wild-type and mutant bovine pancreatic trypsin inhibitor in aqueous solution at 4-80 degrees C. These data lead to the first determination of the exchange rate of a water molecule buried in a protein. The strong temperature dependence of this rate is ascribed to large-scale conformational fluctuations in an energy landscape with a statistical ruggedness of approximately 10 kJ mol(-1).

Using magnetic relaxation dispersion (MRD), we have previously shown that the four internal water molecules in bovine pancreatic trypsin inhibitor (BPTI) exchange with bulk water on time scales between 10(-8) and 10(-4) s at room temperature. Because this exchange is controlled by the protein structure, internal water molecules can be used to probe rare conformational fluctuations. Here, we report (2)H and (17)O MRD data at three temperatures for wild-type BPTI and two BPTI variants where the 14-38 disulfide bond has been cleaved by a double Cys --> Ser mutation or by disulfide reduction and carboxamidomethylation. The MRD data show that the internal water molecules are conserved on disulfide cleavage. However, the exchange rate of the water molecule buried near the disulfide bond is enhanced by 2-4 orders of magnitude. The relation of water exchange to other dynamic processes in BPTI is discussed.