NMR Analyses of Kinase Mutants

Intensity retention experiments were performed on ILV-labeled kinases using a Bruker AVANCE NEO NMR spectrometer operating at a ¹H frequency of 800 MHz equipped with a TCI cryogenic probe. ¹H/¹³C HMQC spectra were collected at 25 °C on 50 μM and 250 μM samples of WT^0Y, V564I^0Y, and V564E^0Y in 25 mM HEPES pH 7.5, 150 mM NaCl, and 40 mM MgCl₂ in the presence and absence of 20 mM ATP. Intensity retention values were calculated as the peak intensity in the ATP/Mg²⁺-bound state divided by the intensity in the absence of ATP. ¹H/¹³C HMQC spectra for 50 μM samples in the absence of ATP were collected with eight scans, while spectra in the presence of 20 mM ATP were collected with 12 scans to account for the dilution upon nucleotide addition (50 μL nucleotide into 200 μL kinase sample). ¹H/¹³C HMQC spectra for 250 μM samples in the presence and absence of ATP were each collected with four scans. To compensate for the dilution factor and/or scan difference in HMQC spectra, peak heights of kinase samples at 250 μM in the presence of ATP were multiplied by 1.25, while peak heights of kinase samples at 50 μM in the absence of ATP were multiplied by 1.2. The peak height multiplications were done in an identical manner for WT^0Y, V564I^0Y, and V564E^0Y.