Fig. 3: Target validation studies of HpTpx and HpGroEL.
a, Mass shifts identified by IP-MS of HpGroEL and HpTpx modified with Metro (+141 Da) or Metro-P3 (+164 Da) (loss of 31 Da of Metro and Metro-P3 upon reductive activation). b, The proposed covalent adduct formation of 5-nitroimidazoles with cysteines26,27. c, Degrees of modification of HpGroEL with Metro or Metro-P3 determined by IP-MS. Graph shows mean values ± s.d. of three measured IP-MS spectra per condition (n = 3). For 62.5 µM Metro and 250 µM Metro-P3 only two data points could be measured. d, The ATPase activity was determined from the initial slope via linear regression of modified (71% Metro-P3 or 100% Metro) HpGroEL and normalized to unmodified HpGroEL activity. Graph represents mean ± s.d. of 7 experiments (nbio = 7). e, The malachite green assay evaluating HpGroEL activity. A control without HpGroEL was subtracted as baseline. Depicted are mean values of technical triplicates (ntech = 3). f, The DoM of HpTpx with Metro or Metro-P3 determined by IP-MS. Bar charts represent mean values ± s.d. of three measured IP-MS spectra (n = 3). g,h, The peroxidase activity assay of HpTpx modified with Metro (g) or Metro-P3 (h). Rates of peroxide reduction coupled to the Trx system (Trx/Trx reductase) were determined by indirectly monitoring NADPH oxidation. Graph shows initial slopes determined by linear regression. Baseline was subtracted (−HpTpx) before normalization to unmodified HpTpx. Bar charts represent mean values ± s.d. of four (Metro) or five (Metro-P3) replicates (nbio = 4–5). Statistical significance was determined on normalized (d) or unnormalized (g,h) data with one-way ANOVA with multiple comparisons (no correction). P values: >0.05 (n.s.), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
