Topspin version 4

1D and 2D NMR spectra were acquired in deuterated water (Deutero, Kastellaun, Germany) on a Bruker Avance Neo 500 spectrometer equipped with a 5 mm TXI prodigy cryoprobe (Bruker BioSpin GmbH, Rheinstetten, Germany) at 298 K. NMR chemical shifts were reported in parts per million (ppm) relative to the residual solvent signal D₂O at δ 4.79. NMR data were analyzed using Topspin, version 4.06 (Bruker, Biospin GmbH, Rheinstetten, Germany) and ACD/Spectrus Processor 2020.2.1 (ACD/Labs, Toronto, Canada).

Spectra of CYP121A1 were acquired at 25 °C on an Agilent 400 MHz spectrometer with a 30° pulse angle, 1-s delay, and −84 ppm transmitter offset frequency for 10,000 scans per experiment. The temperature was increased in 5 °C increments for the temperature titration experiment, with 2500 scans collected for each sample. The data were processed and analyzed in Bruker, TopSpin, version 4.0.6, with 6-Hz line broadening applied. The binding constants were approximated from NMR titration data with cYY using an equation (Equation 1) adapted from the analysis of chemical-shift perturbations (39 (link)) to instead measure changes in peak intensity for an interaction occurring at a slow chemical exchange. Briefly, observed and maximum chemical-shift perturbations are substituted by observed (ΔI_obs) and maximum (ΔI_max) intensity measurements. Relative peak intensity data were quantified for each titration point at the saturated peak values of −84.22 ppm (dimer) and −85.97 ppm (monomer). The intensity data were plotted against cYY concentrations and the resulting plot fit to Equation 1 in Prism v7.05. $\Delta I_{\text{obs}}=\Delta I_{\max }\left\{\left({\left[P\right]}_t+{\left[cYY\right]}_t+Kd\right)-{\left[{\left({\left[P\right]}_t+{\left[cYY\right]}_t+Kd\right)}^2-4{\left[P\right]}_t{\left[cYY\right]}_t\right]}^{1/2}\right\}/2{\left[P\right]}_t$

CYP121A1 eluted from the Ni–NTA column was diluted to 2 µM in 50 mM TrisHCl, pH 7.4, 300 mM NaCl, and supplemented with 10 mM 3-bromo-1,1,1-trifluoroacetone (BTFA) and 5 mM dithiothreitol (DTT). Protein was incubated overnight at 4 °C without agitation, after which the unreacted BTFA and DTT were removed by gel filtration on a 120 ml bed volume column. 125 nmol of BTFA labeled protein was exchanged into NMR buffer (50 mM potassium phosphate, pH 7.4, 50 mM NaCl, and 10% D₂O) using a 10-kDa molecular mass cut-off filter and adjusted to a final concentration of 250 µM. ¹⁹F-NMR spectra were acquired at 25 °C on an Agilent 400 MHz spectrometer using a 30° pulse angle, a 1 s delay, and − 84 ppm transmitter offset frequency for 10,000 scans. Raw data were processed and analyzed using Bruker, TopSpin version 4.0.6 (https://www.bruker.com/).