Extended Data Fig. 8: Altered cell survival of L. lactis cells expressing LmrP mutants.
From: An embedded lipid in the multidrug transporter LmrP suggests a mechanism for polyspecificity
a, Residues 52 and 56 are positioned within the protein interior, and mutation to tyrosine (as depicted by the placement of a common tyrosine rotamer, yellow spheres) is predicted to perturb the binding of the interior lipid (which is shown by the corresponding 2Fo-Fc map at 1 σ). Bound Hoechst 33342 is shown in stick representation (green). See Fig. 4b for an equivalent panel of residue 116. b, Western blot demonstrating similar levels of expression for wild-type and mutant variants of LmrP. Resuspended membranes were prepared for each mutant at a concentration of 0.2 g /ml in buffer A, as detailed in the methods. Samples of each resuspended membrane were resolved through SDS PAGE followed by Western blot analysis. Target proteins were identified using two-step, indirect detection with murine anti-His used as primary antibodies, and anti-mouse-horseradish-peroxidase (HRP) conjugates used as a secondary antibodies. Antibody-bound proteins were revealed using a chemiluminescent HRP substrate. Uncropped images available as source data online. c, Cell survival MIC50 values (the minimum inhibitory concentration required to inhibit 50% of growth) and corresponding standard error mean, calculated by fitting a sigmoidal dose–response curve on 6 independent curves using Prism 8.0. Resultant graphs from which these are values are derived are shown in Fig. 5b–d.
