Kinetic and thermodynamic analysis of the conformational folding process of SS-reduced bovine pancreatic ribonuclease A using a selenoxide reagent with high oxidizing ability

Redox-coupled folding pathways of bovine pancreatic ribonuclease A (RNase A) with four intramolecular disulfide (SS) bonds comprise three phases: (I) SS formation to generate partially oxidized intermediate ensembles with no rigid folded structure; (II) SS rearrangement from the three SS intermediate ensemble (3S) to the des intermediates having three native SS linkages; (III) final oxidation of the last native SS linkage to generate native RNase A. We previously demonstrated that DHSox, a water-soluble selenoxide reagent for rapid and quantitative SS formation, allows clear separation of the three folding phases. In this study, the main conformational folding phase (phase II) has been extensively analyzed at pH 8.0 under a wide range of temperatures (5–45 °C), and thermodynamic and kinetic parameters for the four des intermediates were determined. The free-energy differences (ΔG) as a function of temperature suggested that the each SS linkage has different thermodynamic and kinetic roles in stability of the native structure. On the other hand, comparison of the rate constants and the activation energies for 3S → des with those reported for the conformational folding of SS-intact RNase A suggested that unfolded des species (desU) having three native SS linkages but not yet being folded are involved in very small amounts (<1%) in the 3S intermediate ensemble and the desU species would gain the native-like structures via X-Pro isomerization like SS-intact RNase A. It was revealed that DHSox is useful for kinetic and thermodynamic analysis of the conformational folding process on the oxidative folding pathways of SS-reduced proteins.

Figure optionsDownload as PowerPoint slideHighlights▸ Three phases of the oxidative folding of RNase A were temporally separated using DHSox. ▸ Thermodynamic and kinetic parameters of the four des intermediates were obtained using DHSox. ▸ Each SS linkage in the native RNase A has different thermodynamic and kinetic roles. ▸ Unfolded des species (desU) gain native-like structures via X-Pro isomerization. ▸ All four des species were isolated and allowed to fold to N under aerobic conditions.