Inova 800 mhz spectrometer

¹⁵N-HSQC spectra were acquired with 0.1 mM ¹⁵N-labeled protein samples in 20 mM sodium sulfate, 150 mM NaCl, 1 mM EDTA, and 1 mM DTT at pH 6.5. NMR titrations were performed by adding unlabeled concentrated RNA (1-5 mM) to ¹⁵N labeled protein (0.1 mM) gradually. NMR spectra were acquired on Varian Inova 800 MHz spectrometer at 293 K.

The ¹H-¹³C HMQC spectra were obtained using Agilent's gChmqc experiment in Biopack (Agilent, Santa Clara, CA). The NMR data were collected on a UNITY INOVA 800 MHz spectrometer equipped with a 5-mm Varian ¹H[¹³C,¹⁵N] triple-resonance cryogenically cooled probe at 25°C or 35°C. In the ¹H dimension, 1024 complex points were acquired with a sweep width of 14 ppm using a relaxation delay of 2 s. In the indirect ¹³C dimension, 96 complex points were acquired with a spectral width of 10 ppm, and the ¹³C offset was set to 13 ppm. A WURST-80 decoupling sequence was used for ¹³C-decoupling during the acquisition period (Kupce and Freeman, 1995 (link)). The residual water peak was suppressed using the WET sequence at the end of the relaxation delay (Smallcombe et al., 1995 (link)). All NMR data were processed by NMRPipe (Delaglio et al., 1995 (link)) and analyzed with NMRViewJ (Johnson and Blevins, 1994 (link)). The NMR samples were concentrated to 270 µL using Amicon ultracentrifugal filters with a 30 kDa cutoff, into the D₂O NMR buffer: 25 mM Tris-HCl-d11, pD 7.5, 50–100 mM KCl, and 10–30 µM DSS as a chemical shift reference.

For the heteronuclear magnetic resonance spectroscopy (NMR) analysis of syntenin-1 and TSPAN6 interaction, uniformly 15 N-labeled syntenin-1 PDZ tandem domain (113 to 273), referred to as “syntenin-1 PDZ12,” was expressed as a GST fusion in Escherichia coli BL21 (DE3) at 25 °C in M9 minimal medium using 15NH4Cl as the sole nitrogen source. The purification of GST-fused protein was performed as reported previously [37 (link)]. The NMR samples contained 200 μM protein, 150 mM NaCl, 500 μM TCEP [Tris(2-carboxyethyl)phosphine], and 50 μM AEBSF in 50 mM Tris buffer (pH 7.5). All NMR spectra were recorded at 30 °C on a Varian Inova 800-MHz spectrometer equipped with a room temperature 5-mm 1H/13C/15 N z-axis pulse field gradient probe, with data processed using the Azara package and analyzed using Ansig [38 (link)]. Synthesized C-terminal TSPAN6 peptide was purchased from Sigma-Genosys and contained an extra Arg residue at the N terminus, to improve peptide solubility. Titrations of syntenin-1 PDZ12 proteins with C-terminal TSPAN6 peptide were conducted by recording a series of 1H,15N HSQC spectra of 15 N-labeled protein (200 μM), with increasing molar concentrations of the peptide ligand, up to a protein-to-peptide ratio of 1:8. The combined backbone 1H and 15 N chemical shift changes, Δδ, were calculated as reported earlier [39 (link)].