Mantatp

All chemical reagents were of the highest purity commercially available. Millipore MiliQ® water (filtered through a 0.2-μm filter) or Sigma RNase-free water was used in all buffers. ATP (Sigma A7699, ≥ 99% purity assayed by HPLC, ≤ 0.1% inorganic phosphate) and ADP (Roche Molecular Biochemicals) concentrations were determined by absorbance using ε₂₅₉ of 15,400 M^− 1 cm^− 1. Polyuridylic acid (polyU RNA) (SC-215733A; Santa Cruz Biotechnology) was dialyzed extensively against distilled deionized water and ethanol precipitated prior to use. RNA concentrations were determined by absorbance using ε₂₆₀ of 9660 M^− 1 cm^− 1 and are in units of nucleotides. Mant-labeled nucleotides (2′ and 3′ mixed isomer, mantATP, and mantADP from Invitrogen;mantATP, mant-dATP, and mant-dADP from Jena Biosciences) concentrations were determined by absorbance (ε₂₅₅ = 23,300 M^− 1 cm^− 1). One molar equivalent of MgCl₂ was added to all nucleotides immediately before use.
All assays were performed at 25 ± 0.1 °C in 30 mM Hepes(pH 7.5), 100 mM KCl, 2 mM MgCl₂, and 2 mM DTT.

Binding of the fluorescent ATP analog 2′-/3′-O-(N-methylanthraniloyl) adenosine 5′-triphosphate (MANT-ATP, JENA biosciences) was detected by titrating PfFlaI (20 nM, 100 nM and 5 µM in buffer A containing 5 mM MgCl₂) with increasing concentrations of MANT-ATP in a 150 µl cuvette at 20 °C in a Fluoromax-4 fluorimeter (HORIBA Scientific, Irvine, CA, USA). Excitation and emission wavelengths were set to 285 and 450 nm respectively with slit widths of 10 nm. Fluorescence was corrected for MANT-ATP fluorescence in the absence of protein. To determine the binding affinity of ATP, competition assays between MANT-ATP and ATP were performed. Total fluorescence was determined under the conditions described above after addition of increasing amounts of ATP to a solution containing 20 nM PfFlaI and 10 nM of MANT-ATP. The data were fitted with the Hill equation: $F=\left(F_{\max }+\left(F_{\min }-F_{\max }\right)\ast {\left[\text{ATP}\right]}^n\right)∕\left({\text{IC}}_{50}^n+{\left[\text{ATP}\right]}^n\right)$ , where F = Fluorescence, F_min = minimal fluorescence, F_max = maximal fluorescence, [ATP] is the ATP concentration, IC₅₀ is the ATP concentration where the fluorescence is reduced by half, and n = Hill coefficient.

A 1 mM stock solution of the fluorescent mant-ATP (2′/3′-(N-methyl-anthraniloyl)-adenosine-5′-triphosphate triethylam-monium salt; Jena Bioscience) was prepared in buffer D and titrated with EssC-C. Fluorescence was monitored at 20°C using a LS44 spectrometer (PerkinElmer) with an excitation wavelength of 350 nm (or 360–550 nm for the emission spectrum) and an emission wavelength of 448 nm at a bandwidth of 5 nm. Bound mant-ATP was displaced by titration with 100 mM ATP.

FRET using intrinsic tryptophan fluorescence is used to monitor mant-ATP (obtained from Jena Bioscience) binding kinetics to Aurora A at 10°C. In the binding experiment or $k_{on}$ , increasing concentration of mant-ATP were quickly mixed to 0.5 µM Aurora A (ratio 1:10, excitation at 295 nm, emission cut-off at 395 nm). In the experiment to measure the release of mant-ATP or $k_{off}$ , 10 µM/10 µM Aurora A/mant-ATP complex was diluted with buffer (ratio 1:10). A significant decrease in the fluorescence intensity of Aurora A (excitation at 295 nm, emission cut-off at 395 nm) can be seen due to the mant-ATP release.

Binding of MANT-ATP and MANT-ATPγS to different full-length RIG-I mutants (1–925, N-terminal His-tag) was determined via Förster resonance energy transfer from RIG-I to MANT-ATP (Jena Bioscience, Jena, Germany). All samples were prepared in 96-well black chimney microplates. 2.2 µM protein and equimolar concentrations of a 14mer dsRNA (5'-CGACGCUAGCGUCG-3', see 'Protein crystalization') were pre-incubated with different MANT-ATP concentrations (0.2 µM – 200 µM) for 15 min at room temperature in assay buffer (25 mM HEPES, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT, pH 7). Fluorescence of MANT-ATP was measured in a TECAN M1000 microplate reader at λ_ex/_em = 290/448 nm, gain = 170 using an average of five reads per well. Assays were performed four times in independent experiments using the same protein purification batch. For determination of affinities fluorescence values were fitted to Equation (1) using R, where y is the fluorescence, [P] is the protein concentration, B_max is the maximal fluorescence and K_D is the dissociation constant. Fitting was performed globally on all available datasets. Representative values in figures are mean values ± SD.