Cryogenic triple resonance probe

For the medium analysis, 450 μL of medium was supplemented with 50 μL of 10x D₂O NMR buffer (50 mM NaH₂PO₄, 20 mM Na₂HPO₄, 0.25% TSP). For the extracted metabolites, the aqueous layer was dissolved in 1x D₂O NMR buffer (5 mM NaH₂PO₄, 2 mM Na₂HPO₄, 0.025% TSP in 100% D₂O). The lipid-soluble layer was dissolved in 100% CDCl₃. The samples were transferred to a 5 mm NMR tube. The 2D-HSQC NMR spectra were obtained using an 800 MHz Bruker Avance spectrometer equipped with a cryogenic triple-resonance probe (Bruker, Billerica, MA, USA). The HSQC spectra were obtained with a Bruker pulse sequence hsqcetgpsisp2.2. The spectral widths were set to 16 ppm (¹H) and 40 ppm (¹³C), with 1024 (T2) × 120 (T1) complex points and 4 scans per increment.

The ¹⁵N- and ¹³C-labeled SsoSSB_1–114 sample was dissolved to a final protein concentration of 0.5–1.2 mM with 10% D₂O in buffer A. A Bruker 900 MHz NMR spectrometer equipped with a cryogenic triple-resonance probe at the Korea Basic Science Institute (Ochang, Korea), Bruker AVANCE Neo 600 MHz spectrometers at GIST Central Research Facilities with a cryogenic triple-resonance probe (Gwangju, Korea), and an Agilent DD2 700 MHz NMR spectrometer at Gyeongsang National University (Jinju, Korea) were used to collect NMR spectra. Backbone and sidechain assignments were performed in previous studies [28 (link),66 (link)]. ¹⁵N- and ¹³C-edited NOESY-HSQC were collected at 50 °C with 150 ms and 300 ms mixing times for structure calculation. In CSP experiments, ssDNA composed of 15 adenines (dA(15)) was added at molar ratios ranging from 0:1 to 2:1 to ¹⁵N-labeled SsoSSB_1–114. Average CSP values (Δδ_avg) were calculated using the following equation
$${\mathsf{\Delta }\mathsf{\delta }}_{\mathrm{avg}}=\sqrt{{(\frac{\mathsf{\Delta }\mathsf{\delta }\mathrm{N}}{5.88})}^2+{\left(\mathsf{\Delta }\mathsf{\delta }\mathrm{H}\right)}^2}$$

Six plates of 70% confluent cultured cells were harvested with centrifugation. After re-suspending the cells with 5 ml DPBS, the cells were counted and 3 × 10⁷ cells were transferred into a fresh tube. After centrifugation, the harvested cells were re-suspended in 500 μl glucose-free DMEM media (Gibco) supplemented with 10% dialyzed FBS, 25 mM U-¹³C₆-labeled glucose, and 10% D₂O. The cells were spun in an NMR (30 g, 100 s) to allow sedimentation, enough to cover the active region of the NMR detection coil. ¹H-¹³C Heteronuclear Single Quantum Coherence (HSQC) NMR spectra were measured using the 800-MHz Bruker Avance spectrometer equipped with a cryogenic triple resonance probe (Seoul National University). The dataset comprises 1024 × 128 points for direct and indirect dimensions, respectively. The time course spectral measurement was obtained at 310 K for 24 time points, with each experiment lasting for 288 s (13 points). Each metabolite was identified by spiking the standard compound.

Standard ¹⁵N-¹H HSQC experiments were carried out at a ¹H frequency of 700 MHz. Spectra of ¹⁵N-labeled C4 scFv were recorded free or in the presence of 1.2 molar equivalents of unlabeled HTT(1-17) peptide (Genemed Synthesis Inc., New York, USA). All experiments were recorded in 10 mM phosphate and 150 mM NaCl at pH 5.9 and at 310 K. For backbone assignments of C4 scFv in its unbound state, standard triple resonance experiments [HNCA, CBCA(CO)NH, HNCACB, HNCO and HN(Ca)CO spectra] were recorded at 310 K using a Bruker Avance 700-MHz spectrometer equipped with a cryogenic triple resonance probe (Bruker, Coventry, UK). The chemical shifts of individual spin systems (HN, N, Ca, Cb and CO) were collected manually and the backbone resonance assignments were achieved iteratively through a combination of computer-aided automated assignment procedure using the program Mars [65] (link). HNCA and CBCA(CO)NH spectra were recorded to determine the assignments of the resonances of the N, H, Ca and Cb atoms of the residues of C4 scFv in the presence of an equimolar amount of unlabeled HTT(1-17) peptide.

R₁ and R₂ of ¹⁵N, and ¹H-¹⁵N hetNOE data, were recorded on the Bruker AVANCE Neo 600 MHz spectrometers at GIST Central Research Facilities with cryogenic triple-resonance probes (Gwangju, Korea). Pseudo-3D NMR spectra were collected with relaxation delays of 20, 60, 100, 200, 400, 600, 800, 1000, 1200, and 1600 ms at 25 °C and 50 °C for the ¹⁵N R₁ measurements, and with relaxation delays of 16.96, 33.92, 67.84, 101.76, 135.68, 203.52, 271.36, 339.2, 407.04, and 547.72 ms at 25 °C and 50 °C for the ¹⁵N R₂ measurements. POKY was used to extract the relaxation rate constants by fitting the decay of peak height as a function of the relaxation delay to a single exponential function [38 (link)]. For the hetNOE measurement, interleaved 2D ¹H-¹⁵N HSQC spectra were acquired with and without an initial proton saturation of 2.5 s at 25 °C and 50 °C. hetNOE values were obtained from the ratios of peak heights between pairs of spectra, calculated with a POKY script [38 (link)]. For more accurate analysis, overlapping peaks were excluded from the data. The rotational correlation time (τ_c) was calculated by this equation [39 (link),40 (link)]
$${\mathsf{\tau }}_c=(\frac{1}{4{\mathsf{\pi }\mathsf{\nu }}_N})\sqrt{(6\frac{R_2}{R_1}-7)}$$
where ν_N is the resonance frequency of ¹⁵N in Hz.

NMR experiments were performed at 298 K, on Bruker 800 MHz spectrometers equipped with cryogenic triple-resonance probes. Typically, NMR samples contained 0.2 ml of up to 50/100 µM protein in 50 mM Tris pH 8.3, 500 mM NaCl, 2 mM TCEP, 1 mM TMSP, 5% D₂O. The spectra were processed and analysed using Topspin 3.5 (Bruker Biospin). For acquiring the ¹H, ¹³C/¹⁵N-HSQC spectra of the FXR –peptide (NCoA or NCoR) complex, a ratio of 1:1 (50 µM protein; 50 µM peptide) was used for the measurements. The direct interaction of ligands (agonist/antagonist/partial agonist) and their influence on the binding of the co-activator/co-repressor binding was monitored by measuring ¹H/¹³C-HSQC spectra of the ¹³C-¹⁵N-Arg/Leu selectively labeled peptides (ratio: Protein:peptide:ligand was 50:50:250 µM) in the apo and in complex with protein. Samples were measured in a 3 mm NMR-tube with a sample volume of 0.2 mL. FXR was at a concentration of 50 µM and the peptide and ligands were added to a final concentration of 50 µM (from a stock solution of 1 mM in water; the stock solutions were preheated to 60–70 °C for 10 min to dissolve any aggregates) and 250 µM (from a stock solution of 100 mM in d₆-DMSO), respectively. (Note: most ligands were not completely soluble. The insoluble precipitate of the excess ligand was spun down and the clear sample solution was then used for NMR measurements).

For NMR experiments, samples were concentrated to 250–400 µM before addition of 10 mM ADP and 10% v/v D₂O and 0.15 mM DSS (final concentrations). NMR spectra of isotope labeled BmrA-NBD in 50 mM BisTris pH 7, 50 mM NaCl were recorded at 298 K on Bruker AVANCE 600, 800, and 900 MHz spectrometers equipped with cryogenic triple resonance probes (Bruker GmbH, Karlsruhe, Germany). TROSY-based ¹⁵N-HSQC, HNCA, HNCO, HN(CA)CO, HN(CO)CA, HNCACB and HNCOCACB experiments were recorded using standard pulse sequences (Salzmann et al. 1998 (link)). All spectra were processed using Bruker TOPSPIN 4.0.8 and analyzed using CARA (Keller 2004 ).
The secondary structure of WT BmrA-NBD based on the backbone chemical shift assignment for the ADP-bound state was determined with TALOS-N (Shen and Bax 2013 (link)).