Fig. 5: Biophysical characterization of small molecule ATUX-8385 binding with protein PR65.
From: Direct observation of small molecule activator binding to single PR65 protein
a NanoDSF traces of the thermal denaturation of PR65 in the absence (green traces) and in the presence of ATUX-8385 (orange traces). The data shown are the first derivative of the ratio of fluorescence intensities read at 350 nm and 330 nm (dFIR (350 nm/330 nm)). The global minimum corresponds to the melting temperature of the protein, Tm. A shift towards higher Tm values indicates an increase in stability induced upon SMAP binding. b Extracted Tm values from the NanoDSF traces indicate an upwards shift in the presence of ATUX-8385(Tm PR65 = 52.7 ± 0.1 °C, Tm PR65+ATUX-8385 = 53.5 ± 0.1 °C, N = 7, p < 0.001 via an unpaired t-test). The NanoDSF experiments were performed with a Prometheus NanoDSF instrument (NanoTemper Technologies). 2 μM of PR65 in PBS, 2 mM DTT, was incubated either with 10% DMSO, or 100 μM ATUX-8385 in a final concentration of 10% DMSO, and thermal denaturation was performed from 20 °C to 90 °C with a 1 °C/min rate. c 19F NMR recorded at 298 K on ATUX-8385 indicates binding of the small molecule to PR65. The NMR spectra show a drop in signal intensity upon binding of the small molecule, while the change in chemical shift is small as indicative of relatively weak binding. 19F 1D CPMG experiments were performed for 100 μM ATUX-8385 in 10% DMSO (blue: transverse period 2 ms; cyan: transverse period 102 ms) or for 100 μM ATUX-8385 in 10% DMSO with 5 μM PR65 (red: transverse period 2 ms; magenta: transverse period 102 ms) on a Bruker Avance III 600 MHz (19F 564 MHz). d Fluorescence polarization experiments show ATUX-8385 binding on PR65 with a KD in the low micromolar range. Shown are the fluorescence polarization values (mP) for 2.5 μM, 5 μM, and 10 μM ATUX-8385 upon PR65 titration, N = 3. One-site fitting of the data (see methods for equation) gives KD = 9.4 ± 1.4 μM.
