Reductive cleavage and regeneration of the disulfide bonds in Streptomyces subtilisin inhibitor (SSI) as studied by the carbonyl 13C NMR resonances of cysteinyl residues.
Four enhanced carbonyl carbon resonances were observed when Streptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-13C]Cys. The 13C signals were assigned by the 15N, 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.
Uchida
K
K
Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Hachioji-shi, Japan.
Miyake
Y
Y
Kainosho
M
M
D001426:Bacterial Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002247:Carbon Isotopes; D003545:Cysteine / Q000378:metabolism*; D003553:Cystine / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011392:Proline; D011487:Protein Conformation / Q000187:drug effects; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism*; D013302:Streptomyces / Q000737:chemistry / Q000235:genetics; D013381:Subtilisins / Q000037:antagonists & inhibitors*
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D015842:Serine Proteinase Inhibitors; C018150:subtilisin inhibitor protein, Streptomyces; D003553:Cystine; D011392:Proline; D013381:Subtilisins; D003545:Cysteine; D004229:Dithiothreitol