THERMAL-DENATURATION OF RIBONUCLEASE-A CHARACTERIZED BY WATER O-17 AND H-2 MAGNETIC-RELAXATION DISPERSION

Water oxygen-17 and deuteron nuclear magnetic relaxation dispersion (N MRD) measurements were used to characterize ribonuclease A (RNase A) i n the course of thermal denaturation at pH 2 and 4. The structure and dynamics of the protein were probed by specific long-lived water mole cules, by the short-lived surface hydration, and by labile side-chain hydrogens. The NMRD data show that native RNase A contains at least th ree water molecules with a mean residence time of 8 ns at 27 degrees C and an activation enthalpy of ca. 40 kJ mol(-1). These water molecule s are identified with some or all of six ordered water molecules partl y buried in surface pockets in the crystal structure of RNase A. The l oss of the O-17 dispersion at higher temperatures demonstrates that, i n the thermally denatured protein, these surface pockets are either no t present or undergoing large structural fluctuations on a subnanoseco nd time scale. The relaxation dispersion step vanishes monotonically a nd essentially in concert with the CD denaturation curves, thus ruling out the existence of equilibrium intermediates with a substantial amo unt of non-native and long-lived hydration water. The NMRD data show t hat thermally denatured RNase A has a relatively compact but highly fl exible structure. The global solvent exposure and the hydrodynamic vol ume of the denatured protein are much less than for maximally unfolded disulfide-intact RNase A. The NMRD data show that thermal denaturatio n is accompanied by a large reduction of the mean-square orientational order parameter of side-chain O-H bonds, implying that, in the denatu red state, these side chains sample a wide distribution of conformatio nal states on a subnanosecond time scale.