Refolding of Thermally and Urea-Denatured Ribonuclease A Monitored by Time-Resolved FTIR Spectroscopy

Abstract

We undertook a first detailed comparative analysis of the refolding kinetics of ribonuclease A (RNase A) by time-resolved Fourier transform infrared spectroscopy. The refolding process was initiated either by applying a temperature jump on the thermally denatured protein or by rapid dilution of a concentrated [¹³C]urea solution containing the chemically unfolded protein. The dead time of the injecting and mixing devices and the time-resolution of the spectrometer permitted us to monitor the refolding kinetics in a time range of 100 ms to minutes. The infrared amide I‘ band at 1631 cm^-1 was used to directly probe the formation of β-sheet structure during the refolding process. The aromatic ring stretching vibration of tyrosine at 1515 cm^-1 was employed as a local monitor that detects changes in the tertiary structure along the folding pathway. The comparative analysis of the kinetics of the β-sheet formation of chemically and thermally denatured ribonuclease A revealed similar folding rates and amplitudes when followed under identical refolding conditions. Therefore, our kinetic infrared studies provide evidence for a high structural similarity of urea-denatured and heat-denatured RNase A, corroborating the conclusions derived from the direct comparison of the infrared spectra of thermally and chemically denatured RNase A under equilibrium conditions [Fabian, H., & Mantsch, H. H. (1995) Biochemistry 34, 13651−13655]. In detail, the kinetic infrared data demonstrate that in the time window of 0.1−30 s approximately 40% of the native β-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 the β-structure is formed out of the time window used in this study. Temperature jump experiments in the absence of chemical denaturants exhibited faster and more complex refolding kinetics. In addition, differences in the time constants of refolding derived from the amide I‘ band at 1631 cm^-1 and from the tyrosine vibration at 1515 cm^-1 were observed, indicating that the formation of secondary structure precedes the formation of stable tertiary contacts during the refolding of RNase A.