SPR competition assays

SPR competition assays were performed by using a BIAcore S200 instrument and the A-B-A inject function (62). Competition A-B-A analyses were used to interrogate specificity of the potential ligand binding site preferences of Tlp10 and to unravel the nature of the ligand-sensor interactions. This assay was designed to show if a cumulative response is observed when a second analyte (B) is flown across the bound protein saturated with the first analyte (A) (Fig. 4 and table S6). As the assay is designed to provide saturation of all analytes tested, this assay does not provide 1:1 competition to indicate which is the preferred analyte for a binding site. The proteins, Tlp10^LBD wild type, Tlp10^LBD Y70A, and Tlp10^LBD N115A, Tlp10^LBD N120A, and Tlp10^LBD H193A mutants were immobilized as above. The A-B-A was used with combinations of each of the compound (at concentration 10 × K_D) and PBS control, with 60 seconds injections of analyte A to ensure saturation or near-saturation was reached prior to competition with analyte B. The results were analyzed using BIAcore S200 evaluation software using the sensorgram mode and data was zeroed to baseline prior to the initial A injection. All response data was normalised to 100Da molecular weight for each analyte allowing direct comparison of responses.