Supplementary Figure 6: Schematic illustrating the experimental design to test for S-subunit exchange by EcfAA′T and calibration of the S-subunit exchange assay with free RibU*.
From: ATP binding drives substrate capture in an ECF transporter by a release-and-catch mechanism
(a) First, a mixture of LmECF–RibU and isolated substrate-bound RibU that has been labeled with a fluorophore (S*) was prepared. Next either ADP, ATP, or no nucleotide is added and the samples are incubated for 10 min at 25 °C. Subsequently, the samples are separated by SEC, and binding of S* by EcfAA′T is detected by monitoring the S* fluorescence relative to elution position. SEC traces of several concentrations of RibU* detected by UV absorbance (b) and TMRM fluorescence(c). The integrated peak areas from (c) were used to calibrate the assay, as shown in (d).
