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 DHS(ox), 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 DHS(ox) is useful for kinetic and thermodynamic analysis of the conformational folding process on the oxidative folding pathways of SS-reduced proteins.
Keywords: 1S, 2S, 3S, and 4S, ensembles of folding intermediates of RNase A with one, two, three, and four SS linkages, respectively; AEMTS, 2-aminoethyl methanethiosulfonate; BPTI, bovine pancreatic trypsin inhibitor; DHSox, trans-3,4-dihydroxyselenolane oxide; DTTox, oxidized DTT; DTTred, dithiothreitol; Disulfide bond; EDTA, ethylenediaminetetraacetic acid; ESI, electron spray ionization; GSSG, oxidized glutathione; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HPLC, high performance liquid chromatography; N, native RNase A; Oxidative protein folding; R, reduced RNase A; RNase A, bovine pancreatic ribonuclease A; Ribonuclease A; SH, thiol; SS, disulfide; Selenoxide; TFA, trifluoroacetic acid; Trans-3,4-dihydroxyselenolane oxide; U, unfolded RNase A; UV, ultraviolet; X-Pro isomerization; desN, folded des intermediate; desU, unfolded des intermediate; des[26–84], des[40–95], des[58–110], and des[65–72], structured 3S intermediates of RNase A having three native SS bonds but lacking one native SS bond specified.