Biochemical characterization of recombinant influenza A polymerase heterotrimer complex: Endonuclease activity and evaluation of inhibitors
Fig 4
Enzymatic characterization of purified proteins.
(A) The endonuclease activity of PA/PB1/PB2 trimer as a function of time and increasing protein concentration. Dilutions of the trimer were prepared in buffer containing 50 mM HEPES (pH 7.5), 100 mM KCl, 1 mM DTT, and 1 mM MnCl2. Reactions were initiated with 1 μM RNA-FRET substrate at room temperature and fluorescence was measured with excitation at 495 nm and emission at 516 nm at 30 second intervals. (B) Different endonuclease activity of the PA/PB1/PB2 trimer in the presence of manganese or magnesium. Dilutions of the trimer were prepared and assayed as described previously using either 1 mM MnCl2 or 5 mM MgCl2. The average reaction rates were calculated from the linear portion of each curve over a 30-minute reaction time and plotted as a function of the trimer enzyme concentration. (C) Determination of kcat for the endonuclease activity of the PA/PB1/PB2 trimer and binding affinity (Km) for the RNA-FRET substrate. 10 nM PA/PB1/Pb2 was incubated in a buffer containing 1 mM MnCl2 as described previously. Two-fold serial dilutions of the RNA-FRET probe from 1 μM were prepared in buffer containing DMSO to 5% final reaction volume. Reactions were initiated with RNA dilutions at room temperature and fluorescence emission was monitored with excitation 495 nm and emission at 516 nm at 30 second intervals. Average rates were calculated from the linear portion of each curve and plotted as a function of RNA concentration. Total hydrolysis of the substrate was achieved by incubation for 10 minutes with 10 U/mL RNAse I and used to generate a standard curve used in the calculation of kcat. The Km and kcat values obtained were 150 ± 11 nM and (1.4 ± 0.2) x 10-3s-1, respectively.