Reductive cleavage and regeneration of the disulfide bonds inStreptomyces subtilisin inhibitor (SSI) as studied by the carbonyl13C NMR resonances of cysteinyl residues |
| |
Authors: | Kenichi Uchida Yoko Miyake Masatsune Kainosho |
| |
Affiliation: | (1) Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Minami-Ohsawa, 192-03 Hachioji-shi, Japan |
| |
Abstract: | Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-13C]Cys. The13C signals were assigned by the15N,13C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys71-Cys101 disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met73-Val74, is more accessible to solvent than the other disulfide bond. Cys35-Cys50, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys71 and Cys101 regenerates at a much faster rate than that between Cys35 and Cys50.Nomenclature of the various states of SSI that are observed in the present study Fully oxidized-folded native or intact (without GuHCl or DTT) - half reduced-folded (Cys71-Cys101 reduced; DTT without GuHCl) - inversely half reduced-folded (Cys35-Cys50 reduced; a reoxidation intermediate from fully reduced-folded state) - fully reduced-unfolded (reduced by DTT in the presence of GuHCl) - fully reduced-folded (an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture) |
| |
Keywords: | Streptomyces subtilisin inhibitor SSI Selective disulfide reduction Dithiothreitol (DTT) Carbonyl13C NMR Double-labeling method Site-specific mutagenesis Stable isotope-assisted NMR Isotope shift DEALS experiment Proline effect |
本文献已被 SpringerLink 等数据库收录! |
|