Fig. 4
Design, validation and regulation of a disulphide-activated DgcB mutant. a Design of the disulfide mutation to lock DgcB into a constitutively active state. S15 and S73 were identified as the best candidates for mutation to cysteine residues as to facilitate the formation of a disulphide bond (a different rotamer for S73, labeled with prime, would be in an appropriate orientation to bond S15 when both mutated to Cys). b Circular dichroism assay with 10 µM protein. Traces ± standard error. Decreasing ellipticity represents increasing c-di-GMP concentration. c Circular dichroism assay with 1 µM of both the single (R218A-S15C only) and double (R218A-S15CS73C) cysteine mutants. The single cysteine mutant has no significant activity compared to the double mutant. d HPLC traces of GTP and c-di-GMP standards, and the end products of the enzyme reactions after 40 min incubation. e HPLC activity assays monitoring c-di-GMP production at 10 µM protein. Bars ± standard deviation. Significance between samples with and without GTP calculated by Student’s t-test (P < 0.001) through GraphPad prism. f HPLC activity assays monitoring c-di-GMP production at 1 µM protein. Significance between R218A-S15C and R218A-S15CS73C calculated by Student’s t-test (P < 0.001) via GraphPad prism. Bars ± standard deviation. g Circular dichroism spectra (reporting on protein folding) of both the reduced and oxidized double mutant (at 0.1 µM protein; signal shown is buffer subtracted). h Circular dichroism c-di-GMP assay of R218A-S15CS73C mutant using 10 µM protein of reduced and oxidized forms (incubated with and without 12.5 mM TCEP overnight). Source data are provided as a Source Data file
