one site – specific binding model of graphpad prism 10  (GraphPad Software Inc)

Journal: Nature Communications

Article Title: A glyoxal-specific aldehyde signaling axis in Pseudomonas aeruginosa that influences quorum sensing and infection

doi: 10.1038/s41467-025-61469-8

Figure Lengend Snippet: A Cell-free supernatants of 24-h cultures of WT MPAO1 harboring either pSB109 empty plasmid control or pSB109-FLAG-ArqI. AqrI expression was induced (+) or not induced (−) with arabinose. B Quantification of pyoverdine and C pyocyanin in supernatants of three biological replicates of experiment shown in A . n = 4 biological replicates. Bars indicate arithmetic mean. Whiskers denote standard deviation. D Thin-layer chromatography (TLC) analysis of extracted PQS from 24-h cultures. Purified PQS (C+) was used as a positive control and standard. E Schematic diagram of the P pqsA -mCherry reporter construct used as a PQS biosensor (this work). F Effect of WT ArqI, ArqI variants, or PA3390 expression on P pqsA -mCherry reporter activity at 24 h post ArqI induction. The y-axis was calculated as relative fluorescent units (RFU) to the empty vector control. n = 3 biological replicates. Bars indicate arithmetic mean. Whiskers denote standard deviation. G Above: Schematic diagram of PqsA fragment (residues 164-413) identified by Y2H as interacting with ArqI. Below: Cartoon representation of the full length PqsA structure generated with the deposited N-terminal domain (PDB code 5OE4; ) and AlphaFold . The ArqI-interacting surface is highlighted in red and substrate shown as blue spheres. The reaction catalyzed by PqsA and ensuing PQS biosynthesis is shown below. H Intrinsic tryptophan quenching of ArqI interacting with PqsA in vitro. Excitation (ex) wavelength set at 295 nm and emission (em) scanned at 320-400 nm range. Plot of changes in tryptophan fluorescence intensities (read at 330 nm) of PqsA full-length protein (FL; top panel) and PqsA N-terminal domain (NTD; bottom panel) titrated with increasing ArqI concentrations. Plots are mean values of 4 trials. K d was determined by fitting in binding saturation: One site – specific binding model of GraphPad Prism 10. Shown are the mean K d ± SEM values. ( I ) One representative of three replicates of dansyl-labeled ArqI quenched by addition of purified PqsA NTD . Fluorescence intensity changes were measured at 512 nm. Curve fitting of these data yielded an average dissociation constant (K d ) of 1.0 ± 0.3 µM ( ± SEM). J Above: Schematic representation of the BioID experiment. Left panel: Western blot analysis of HA-tagged PqsA from P. aeruginosa MPAO1 cell lysate after co-expression of either ArqI-BirA*-FLAG or a BirA*-FLAG control. RpoB was used as a loading control. Right panel: Biotin blot analysis of anti-HA immunoprecipitated (IP) proteins from lysates in the left panel. Representative blots shown of three independent experiments. For panels B , C , and F : * P < 0.05; ** P < 0.01; *** P < 0.001 by two-Way ANOVA with Šídák’s multiple comparisons test ( B , C ) or one-sample t test ( F ). For 4b the p value was calculated as 0.0003 and 4c 0.0065. For 4f (left to right) 0.0052, 0.0008, 0.0031, <0.0001. ArqI contains an N-terminal FLAG tag in A-D, F . PA3390 contains a C-terminal FLAG-tag in F . Source data are provided as a Source Data file.

Article Snippet: K d was determined by fitting in binding saturation: One site – specific binding model of GraphPad Prism 10.

Techniques: Plasmid Preparation, Control, Expressing, Standard Deviation, Thin Layer Chromatography, Purification, Positive Control, Construct, Activity Assay, Generated, In Vitro, Fluorescence, Binding Assay, Labeling, Western Blot, Immunoprecipitation, FLAG-tag