UNFOLDING OF RIBONUCLEASE-A BY GUANIDINIUM CHLORIDE - PROTEIN INTERNAL MOTIONS STUDIED BY NUCLEAR MAGNETIC-RESONANCE SPIN-LATTICE RELAXATION IN AN OFF-RESONANCE ROTATING FRAME

The novel nuclear magnetic resonance (N MR) technique entailing rotating frame spin-lattice relaxation in the presence of an off-resonance radiofrequency field (T1off) has advantageous features for the study of protein molecular motions. Examination of the carbon-13 resonance of the carbonyls in ribonuclease A in the presence and absence of an off-resonance rf field has enabled the overall rotational reorientation time of the enzyme to be determined as 12 ns. The technique also is used to investigate the local mobility of different moieties of ribonuclease A as it is unfolded by the sequential addition of guanidinium chloride. Specifically, the resonances of the C2 protons of the histidine residues are monitored with the experimental variable being the ratio of a peak intensity in the presence to that in the absence of an rf field applied off-resonance. The intensity ratio is related to the mobility of the residue. It is found that the mobility increases for His-12 and His-105 as guanidinium chloride is added to the ribonuclease solution (1.0 mM, pH 5.4) up to a concentration of 1.1 M, which is much below the concentration required for complete unfolding of the protein. No further significant changes in the mobility of His-i 2, His-105, and His-119 are detected as guanidinium chloride is added up to a concentration producing total unfolding of all the ribonuclease. At guanidinium chloride concentrations above 2M, a histidine C2 proton resonance corresponding to the cross-linked random coil protein appears which behaves in the presence of the off-resonance rf field as originating from a residue with greater motional freedom than the histidines on the folded protein. As guanidinium chloride is added to the enzyme in concentrations greater than 3 M, the T1off experiment indicates that the mobility of His-48 is increased considerably. Use of the T1off technique, thus, has permitted the discovery of an intermediate with altered local motion in the ribonuclease unfolding process. Previous studies2 found no change in the spin-lattice relaxation time (T1) for any of the histidine C2 protons of ribonuclease as guanidinium chloride concentration was increased. © 1979, American Chemical Society. All rights reserved.