Advance 600 mhz spectrometer

In the saturation transfer difference (STD) NMR experiment, the entire TlpA_LBD protein was first saturated at the protein resonances, and excess ligand was then added. As the ligand binds and releases from the receptor, saturation transfers from the protein to the bound ligand. This transfer appeared as an increase in the ligand intensity on epitopes that interacted with the TlpA_LBD protein. For STD NMR experiments, samples of 25 μM TlpA_LBD in complex with either 2.5 mM arginine (Arg) or fumaric acid (Fum) in 99% D₂O were prepared. All STD NMR spectra were acquired in Shigemi tubes (Shigemi, USA) with a Bruker 600-MHz Advance spectrometer at 283 K using a 1^H-13^C-15^N gradient cryoprobe equipped with z-gradients. Protein resonances were saturated at −1.0 ppm (on-resonance) and 33 ppm (off-resonance), with a total saturation time of 2 s. A total of 512 scans per STD NMR experiment were acquired, and a Watergate sequence was used to suppress the residual HDO signal. A spin-lock filter with a 5-kHz strength and a duration of 10 ms was applied to suppress the protein background. On- and off-resonance spectra were stored and processed separately, and the final STD NMR spectra were obtained by subtracting the on- and off-resonance spectra. Control STD NMR experiments were performed identically in the absence of protein.

The intracellular dried samples were resuspended in 700 μL phosphate buffer (0.2 M Na₂HPO₄/0.04M NaH₂PO₄, pH 7.4) prepared in a mixture of H₂O/D₂O (80:20; v:v). The samples were centrifuged at 13,000 g for 10 min. 50 μL of a 7 mM solution of 3-trimethylsilyl propionic-2,2,3,3-d4 acid (TSP) reference prepared in 100% deuterium oxide was added to 650 μL of each supernatant. Finally, 700 μL of this final mixture was transferred into 5 mm NMR tubes prior to analysis. For the culture media preparation, 500 μL of each sample were mixed with 250 μL of phosphate buffer. The samples were prepared as described above, with an addition of 14 mM TSP solution. Acquisition of the ¹H-NMR spectra were processed on a Bruker 600-MHz Advance spectrometer for ¹H, using the NOESYPRESAT-1D pulse sequence. The same acquisition method was applied to the samples of cellular extracts and culture media using 256 scans.

¹H NMR spectroscopy was used to detect the chemical environment of the hydrogen atoms of the drug. The samples were dissolved in deuterated dimethyl sulfoxide as described above, placed in an NMR tube, and analyzed using an Advance 600 MHz spectrometer (Bruker, Benxi, China).