RESIDENCE TIMES OF THE BURIED WATER-MOLECULES IN BOVINE PANCREATIC TRYPSIN-INHIBITOR AND ITS G36S MUTANT

The three-dimensional structure of the bovine pancreatic trypsin inhibitor (BPTI) contains 4 internal water molecules, denoted W111, W112, W113, and W122, the latter being replaced by a seryl side chain in the BPTI(G36S) analogue. To investigate the effect of the exchange between these explicit water sites and the hulk solvent, we have measured water O-17 and H-2 nuclear magnetic relaxation in solutions of BPTI and the G36S mutant over the Larmor frequency range 2.6-49 MHz. A comparison of the data from the two nuclei shows unequivocally that the isolated buried water molecule, W122, of BPTI contributes only to H-2, but not to O-17 relaxation, while the other 3 waters contribute fully to the relaxation of both nuclei. This demonstrates that the residence time of W122 is in the range 10-200 mu s, while the residence times of W111-W113 are in the range 15 ns(-1) mu s. The slower exchange of W122 indicates that the functionally active region of BPTI, near the Cys14-Cys38 disulfide bond, is less flexible than the central region of BPTI, where the other 3 buried waters are located.