Journal o1" Biomolecular NM R, 1 ( 1991 ) 49 64 49 ESCOM J-Bio NMR 003 Reductive cleavage and regeneration of the disulfide bonds in Streptomyces subtilisin inhibitor (SSI) as Studied by the carbony113C NMR resonances of cysteinyl residues Kenichi Uchida, Yoko Miyake and Masatsune Kainosho* Department of Chemi.~try. Faculty ~f Science. Tokyo Metropolitan University. Minami-Ohsawa. Hachiqji-shi. 192-03 Japan Received 3 December 1990 Accepted 8 January 1991 Keywor~L~v Streptomyces subtilisin inhibitor: SSl: Selectivedisulfide reduction: Dithiothreitol (DTT); Carbonyl ~C NMR; Double-labeling method: Site-specificmutagenesis: Stable isotope-assisted NMR: Isotope shift: DEALS experiment: Proline effect SUMMARY Four enhanced carbonyl carbon resonances were observed when Streptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [l-13C]Cys. The L~C signals were assigned by the LSN, I-~C 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 ofdithiothreitol (DTT). The re- sults indicate that, in the absence of denaturant, the CysT~-Cys "~l disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond MetTLVal TM, is more accessible to solvent than the other disulfide bond, Cys-~5-Cys -~~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 hydrochlo- ride (GuHCI), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully re- duced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evi- denced by the Cys carbonyl carbon chemical shifts, upon removing GuHC1 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 Cys 7~ and Cys ~~ regenerates at a much faster rate than that between Cys 3-~ and Cys 5~ Nomenclature r~/ the various states ~["SSI that are observed in the present study." Fully o.xidized~/~ihh, d = native or inlact (without GuHCI or DTT): halfre~htced-/~hled (CysTI-Cys I~ reduced: DTT without GuHCI): inversely hal/reduced-/ohh, d (Cys~-Cys ~j reduced: a reoxidation intermediate from fully reduced-folded state): .lidO' reduced-ut!/blded (reduced by DTT in the presence of GuH CIJ:./id/y rethu'ed-/~Jhh'd (an intermediate state obtained by removing DTT and GuHCI from the fully reduced-unfolded SSI reaction mixture). * To whom correspondence should be addressed. 0925-2738 $ 5.00 ~ 1991 ESCOM Science Publishers B.V.