NMR Spectroscopy-Based ATP Binding Kinetics

ATP titrations were performed at 25 °C on WT^0Y, V564I^0Y, and V564E^0Y using a Bruker AVANCE NEO NMR spectrometer operating at a ¹H frequency of 800 MHz equipped with a TCI cryogenic probe. ¹H/¹³C HMQC experiments were acquired on ILV-labeled kinases with 4 scans and 60 complex points in the indirect dimension to minimize the experiment time due to ATP hydrolysis. The entire titration was acquired in ~3 h. Titrations were carried out with 50 μM kinase in 25 mM HEPES pH 7.5, 150 mM NaCl, and 40 mM MgCl₂. ATP was titrated at several concentrations ranging from low μM to mM, as displayed in the figure legends. The AMP-PCP titration was performed at 10 °C on FGFR2K using a Bruker AVANCE III NMR spectrometer operating at a ¹H frequency of 600 MHz equipped with a TCI cryogenic probe. ¹H/¹³C HMQC experiments were performed on 533 μM ILV-labeled kinases with two scans and 100 complex points in the indirect dimension. Binding constants were fit using a nonlinear least-squares regression as shown in Eq. 3. $$\begin{array}{c}\text{Δ}{\delta }_{obs} = \text{Δ}{\delta }_{\max }\frac{\left({[P]}_t+{[L]}_t+K_d\right) - \sqrt{{\left({[P]}_t+{[L]}_t+K_d\right)}^2 - 4{\left[P\right]}_t{\left[L\right]}_t}}{2{\left[P\right]}_t}\end{array}$$