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

Abstract

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 ((1)H 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.

Fig. 1.

Downfield region of the ¹H NMR spectra of RNase A unfolded by a 30-fold molar excess of DTT in 0.5 mL 100 mM PBS buffer with a concentration of 1.0–1.1mM at 303K (a) and 313K (b).

Fig. 2.

Kinetic course of Shannon entropy (A), mutual information (B), and correlation coefficient (C) values of ¹H NMR spectra of RNase A reduction by a 30-fold molar excess of DTT in 0.5 mL, 100 mM PBS buffer at 313K with a protein concentration of 1.0–1.1 mM were presented as in the downfield (6.3–10 ppm) region. (Insets) Semilogarithmic plots. (Open circles) Experimental data. (Filled circles) Points obtained by subtracting the contribution of the slow phase from curve (broken lines).

Fig. 2.

Kinetic course of Shannon entropy (A), mutual information (B), and correlation coefficient (C) values of ¹H NMR spectra of RNase A reduction by a 30-fold molar excess of DTT in 0.5 mL, 100 mM PBS buffer at 313K with a protein concentration of 1.0–1.1 mM were presented as in the downfield (6.3–10 ppm) region. (Insets) Semilogarithmic plots. (Open circles) Experimental data. (Filled circles) Points obtained by subtracting the contribution of the slow phase from curve (broken lines).

Fig. 2.

Kinetic course of Shannon entropy (A), mutual information (B), and correlation coefficient (C) values of ¹H NMR spectra of RNase A reduction by a 30-fold molar excess of DTT in 0.5 mL, 100 mM PBS buffer at 313K with a protein concentration of 1.0–1.1 mM were presented as in the downfield (6.3–10 ppm) region. (Insets) Semilogarithmic plots. (Open circles) Experimental data. (Filled circles) Points obtained by subtracting the contribution of the slow phase from curve (broken lines).

Fig. 3.

Kinetic course of Shannon entropy (A), mutual information (B), and correlation coefficient (C) values of ¹H NMR spectra of RNase A during unfolding. Experimental conditions were as for Fig. 2 ▶ except that the temperature was 303K. (Insets) Semilogarithmic plots. (Open circles) Experimental data. (Filled circles) Points obtained by subtracting the contribution of the slow phase from curve (broken lines).

Fig. 3.

Kinetic course of Shannon entropy (A), mutual information (B), and correlation coefficient (C) values of ¹H NMR spectra of RNase A during unfolding. Experimental conditions were as for Fig. 2 ▶ except that the temperature was 303K. (Insets) Semilogarithmic plots. (Open circles) Experimental data. (Filled circles) Points obtained by subtracting the contribution of the slow phase from curve (broken lines).

Fig. 3.

Kinetic course of Shannon entropy (A), mutual information (B), and correlation coefficient (C) values of ¹H NMR spectra of RNase A during unfolding. Experimental conditions were as for Fig. 2 ▶ except that the temperature was 303K. (Insets) Semilogarithmic plots. (Open circles) Experimental data. (Filled circles) Points obtained by subtracting the contribution of the slow phase from curve (broken lines).