Fig. 6: Mutating the distant coevolving residue positions affects the function of other ligand-binding GAF domains.
From: Coevolving residues distant from the ligand binding site are involved in GAF domain function
A Cartoon representation of the miRFP670nano3 GAF domain (PDB: 7LSC) highlighting distant coevolving residues equivalent to PDE5 L267 and F295 (L100 and W125, respectively) and the ligand, biliverdin (BV). B Western blot analysis of the cell lysates prepared from HEK293T cells transfected with either the WT or mutant miRFP670nano3 plasmid constructs and probed using an anti-FLAG antibody showing the expression of biosensor constructs. Whole blot image used for generating the figure is shown in Supplementary Fig. 8. Graphs showing relative fluorescence (C) and bioluminescence (D) of the WT, L100A and W125A mutant miRFP670nano3. Data shown are mean ± S.D. obtained from five independent experiments. The p-values shown in (C) were obtained from Student’s t-test (two-tailed, unpaired, equal variance). For all tests, a p-value of <0.05 was considered statistically significant.
