Fig. 2: The oxidized NmeAcrIIC1 loses its inhibitory activity against Cas9.

a Elution profiles of the SEC binding assay for the reduced NmeAcrIIC1 (blue), NmeHNH (purple), or the two incubated together with twofold excess NmeHNH (red). The concentration for each trace was normalized to 0.1 mM of a single protein. b Elution profiles of the SEC binding assay for the oxidized NmeAcrIIC1 (blue), NmeHNH (purple), or the two incubated together with twofold excess HNH (red). c Fractions from the assays shown in a, b were visualized on a 12% SDS-PAGE gel. Lane 1 to 4 corresponds to reduced NmeAcrIIC1, NmeHNH, the complex fraction from the mixture of reduced NmeAcrIIC1 with twofold excess NmeHNH, and the residual NmeHNH from the mixture above. Lane 5 to 8 corresponds to oxidized NmeAcrIIC1, NmeHNH, the earlier eluent from the mixture of oxidized NmeAcrIIC1 with twofold excess NmeHNH, and the latter eluent from the mixture above. d, e Binding affinities determined by BLI for the reduced NmeAcrIIC1 and the oxidized NmeAcrIIC1 to NmeHNH, respectively. The reduced NmeAcrIIC1 shows a K_d of 1.78 nM, while the oxidized NmeAcrIIC1 gives a very weak response <0.05, indicating very poor binding. f DNA cleavage assays were conducted in the presence of the reduced NmeAcrIIC1 with five-fold excess DTT (relative to Acr) in the buffer, and the oxidized NmeAcrIIC1 without DTT, respectively. Data were replicated independently more than three times with similar results and the representative one is shown. Source data are provided as a Source Data file.