Fig. 5: FRET assay used to continuously monitor His6-CylA-27-412 activity and aSEC data describing the process of inhibitor-induced pro-domain release.
A Structure of the FRET substrate and its cleavage by CylA. Samples of FRET substrate (44 µM) in PBS (pH = 7.4) before and after treatment with His6-CylA-27-412 (200 nM) overnight at 37 °C. B Initial rates of fluorescent peptide formation as a function of initial FRET substrate concentration (2–125 µM). Non-linear concatenate fitting of data from three independent replicates (n = 3) to the Hill form of the Michaelis-Menten model using Origin Pro 2024 gave Km, h and associated errors. Points represent the mean. Error bars represent the standard of deviation and are centered at the mean. C The relationship between relative enzyme activity (vi/vo) and concentration of 9. Relative enzyme activity was determined by monitoring the rate of cleavage of the FRET peptide in the presence of inhibitor (vi) and comparing the rate to an uninhibited control (vo). Non-linear concatenate fitting of data points vi/vo from four independent replicates (n = 4) to a three-parameter function was used to determine IC50. The colored line represents the calculated fit function. Points represent the mean and error bars represent the standard of deviation centered at the mean. D aSEC analysis of SEC-purified His6-CylA-27-412 treated with different concentrations of inhibitor 9. The inhibitor:CylA ratio is indicated next to each trace. E aSEC traces of SEC-purified His6-CylA-27-412 before (black) and after (red) incubation with purified samples of pro-domain. For (D) and (E) absorbance at 220 nm was monitored. Full length versions of the traces featured in (D) and (E) can be found in Supplementary Fig. 7. Statistical significance was calculated using a one sample and one-sided t test. *P < 0.05, **P < 0.01. Source data are provided as a Source Data file.
