Directed evolution provides insight into conformational substrate sampling by SrtA - Fig 3

<p>A) Schematic of the continuous fluorescence assay used to measure the initial acylation kinetics as previously described. B) Schematic of the fluorescence polarization assay used to follow the complete reaction coordinate. The peptide substrate is labeled at the amino terminus with the fluorescent label tetramethylrhodomain (TMR), which is efficiently excited at 557nm and emits at 576nm. In the absence of SrtA or secondary substrate (biotin labeled GGGGGDYK peptide), the fluorescence polarization is low. Alternatively, following the reaction of substrates in the presence of SrtA, avidin binding greatly increases the increases fluorescence polarization. C) Kinetic parameters for wild-type (WT) or SrtA variants.