REFOLDING OF THERMALLY AND UREA-DENATURED RIBONUCLEASE-A MONITORED BYTIME-RESOLVED FTIR SPECTROSCOPY

We undertook a first detailed comparative analysis of the refolding ki netics of ribonuclease A (RNase A) by time-resolved Fourier transform infrared spectroscopy. The refolding process was initiated either by a pplying a temperature jump on the thermally denatured protein or by ra pid dilution of a concentrated [C-13]urea solution containing the chem ically unfolded protein. The dead time of the injecting and mixing dev ices and the time-resolution of the spectrometer permitted us to monit or the refolding kinetics in a time range of 100 ms to minutes. The in frared amide I' band at 1631 cm(-1) was used to directly probe the for mation of beta-sheet structure during the refolding process. The aroma tic ring stretching vibration of tyrosine at 1515 cm(-1) was employed as a local monitor that detects changes in the tertiary structure alon g the folding pathway. The comparative analysis of the kinetics of the P-sheet formation of chemically and thermally denatured ribonuclease A revealed similar folding rates and amplitudes when followed under id entical refolding conditions, Therefore, our kinetic infrared studies provide evidence for a high structural similarity of urea-denatured an d heat-denatured RNase A, corroborating the conclusions derived from t he direct comparison of the infrared spectra of thermally and chemical ly denatured RNase A under equilibrium conditions [Fabian, H., & Mants ch, H. H. (1995) Biochemistry 34, 13651-13655]. In detail, the kinetic infrared data demonstrate that in the time window of 0.1-30 s approxi mately 40% of the native beta-sheet structure in RNase A is formed in the presence of 0.6 M urea at pH 3.6, indicating that up to 60% of th e beta-structure is formed out of the time window used in this study. Temperature jump experiments in the absence of chemical denaturants ex hibited faster and more complex refolding kinetics. In addition, diffe rences in the time constants of refolding derived from the amide I' ba nd at 1631 cm(-1) and from the tyrosine vibration at 1515 cm(-1) were observed, indicating that the formation of secondary structure precede s the formation of stable tertiary contacts during the refolding of RN ase A.