Thermal denaturation of ribonuclease A characterized by water 17O and 2H magnetic relaxation dispersion.
Water oxygen-17 and deuteron nuclear magnetic relaxation dispersion (NMRD) measurements were used to characterize ribonuclease A (RNase A) in the course of thermal denaturation at pH 2 and 4. The structure and dynamics of the protein were probed by specific long-lived water molecules, by the short-lived surface hydration, and by labile side-chain hydrogens. The NMRD data show that native RNase A contains at least three water molecules with a mean residence time of 8 ns at 27 degreesC and an activation enthalpy of ca. 40 kJ mol-1. These water molecules are identified with some or all of six ordered water molecules partly buried in surface pockets in the crystal structure of RNase A. The loss of the 17O dispersion at higher temperatures demonstrates that, in the thermally denatured protein, these surface pockets are either not present or undergoing large structural fluctuations on a subnanosecond time scale. The relaxation dispersion step vanishes monotonically and essentially in concert with the CD denaturation curves, thus ruling out the existence of equilibrium intermediates with a substantial amount of non-native and long-lived hydration water. The NMRD data show that thermally denatured RNase A has a relatively compact but highly flexible structure. The global solvent exposure and the hydrodynamic volume of the denatured protein are much less than for maximally unfolded disulfide-intact RNase A. The NMRD data show that thermal denaturation is accompanied by a large reduction of the mean-square orientational order parameter of side-chain O-H bonds, implying that, in the denatured state, these side chains sample a wide distribution of conformational states on a subnanosecond time scale.
Denisov
V P
VP
Condensed Matter Magnetic Resonance Group, Department of Chemistry, Lund University, Sweden.
Halle
B
B
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010103:Oxygen Isotopes; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D012996:Solutions; D012997:Solvents; D013816:Thermodynamics; D014867:Water / Q000737:chemistry* / Q000378:metabolism
D010103:Oxygen Isotopes; D012996:Solutions; D012997:Solvents; D014867:Water; D003903:Deuterium; D012259:Ribonuclease, Pancreatic