Identification of formation of initial native structure in onconase from an unfolded state

摘要

In the oxidative folding of onconase, the stabilization of intermediates early in the folding process gives rise to efficient formation of its biologically active form. To identify the residues responsible for initial formation of structured intermediates, the transition from an ensemble of unstructured three-disulfide species, 3SU, to a single structured three-disulfide intermediate species, des-[30–75] or 3SF, at pH 8.0, 25 °C was examined. This transition was first monitored by far-UV CD spectroscopy at pH 8.0, 25 °C, showing that it occurs with formation of secondary structure, presumably due to native interactions. The time-dependence of formation of native-like structure was then followed by NMR spectroscopy after arresting the transition at different times by lowering the pH to 3 and then acquiring ¹H,¹⁵N – HSQC spectra at pH 3, 16 °C to identify amide hydrogens that become part of native-like structure. H/D exchange was utilized to reduce the intensity of resonances from backbone amide hydrogens not involved in structure, without allowing exchange of backbone amide hydrogens involved in initial structure. Six hydrogen-bonding residues, namely, Tyr38, Lys49, Ser82, Cys90, Glu91, and Ala94 were identified as involved in the earliest detectable native-like structure before complete formation of des-[30–75], and are further stabilized later in the formation of this intermediate through S-S/SH interchange. By observing the stabilization of the structures of these residues by their neighboring residues, the initial, native-like structural elements formed in this transition have been identified, providing details of the initial events in the oxidative folding of onconase.