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Two-phase unfolding pathway of ribonuclease A during denaturation induced by dithiothreitol

¹ Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, China
² National Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing 100084, China

Reprint requests to: Professor Ri-Qing Zhang or Professor Hai-Meng Zhou, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China; e-mail: zhangrq{at}mail.tsinghua.edu.cn; zhm-dbs{at}mail.tsinghua.edu.cn; fax: 86-10-62785505.

The dynamics of the unfolding process of bovine pancreatic ribonuclease A (RNase A) unfolded by dithiothreitol (DTT) at a low concentration of 1:30 were investigated in alkaline phosphate-buffered saline solutions at 303K and 313K by using proton nuclear magnetic resonance (¹H NMR) spectra. Three NMR spectral parameters including Shannon entropy, mutual information, and correlation coefficient were introduced into the analysis. The results show that the unfolding process of RNase A was slowed to the order of many hours, and the kinetics of the unfolding pathway described by the three parameters is best fit by a model of two consecutive first-order reactions. Temperature greatly influences the rate constants of the unfolding kinetics with different temperature effects observed for the fast and the slow processes. The consistencies and the differences between the three sets of parameters show that they reflect the same relative denaturation pathway but different spectra windows of the unfolding process of RNase A. The results suggest that the unfolding process of RNase A induced by low concentrations of DTT is a two-phase pathway containing fast and slow first-order reactions.

Keywords: Correlation coefficient; image analysis; mutual information; proton nuclear magnetic resonance; ribonuclease A; Shannon entropy; unfolding dynamics

Abbreviations: C, correlation coefficient • DSS, 2,2-dimethyl-2-sila-pentanesulfonate • DTT, dithiothreitol • FID, free induction decay • H, Shannon entropy • MI, Shannon mutual information • NMR, nuclear magnetic resonance • NOE, nuclear Overhauser enhancement • PBS, phosphate-buffered saline • ppm, parts per million • RNase A, bovine pancreatic ribonuclease A • S/N, signal to noise ratio • 2-D NMR, two-dimensional nuclear magnetic resonance.

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