Data from measurements of fluorescence of BODIPY-GDP bound to EF-G wild type and mutants in the presence of ribosomes with increasing fusidic acid concentration to study build up of fusidic acid stalled complexes.
Reaction components:
500 nM E. coli 70S ribosomes (NEB)
50 nM BODIPY-FL-GDP (Invitrogen)
either 500 nM WT EF-G, 80 nM EF-G H409K/M479E, 2.5 uM EF-G H438P or 5 uM EF-G H439C/G451C
either no FusB or a 5x molar excess FusB with regards to EF-G
Buffer: 50 mM TrisHCl, 70 mM ammonium chloride, 30 mM potassium chloride, 7 mM magnesium chloride, pH 7.5
Reaction temperature: 37C
Fluorescence excitation wavelength 485 nm, emission wavelength 520 nm.
Measured using a FLUROStar Omega plate reader.
All measurements in triplicate.
Increased fluorescence shows increased formation of stalled ribosome:EF-G:GDP:FA complexes. FusB rescues WT and H409K/M479E from inhibition by fusidic acid while stalled complexes are still formed in presence of FusB for H438P and H438C/G451C, suggesting FusB is less able to protect these mutants from fusidic acid inhibition.
