700 mhz spectrometer

NMR samples were prepared at a concentration of approximately 1 mM in 0.6 mL (H₂O/D₂O 9:1 v/v) of buffer solution with 10 mM KH₂PO₄/K₂HPO₄, 70 mM KCl, and 0.2 mM EDTA (pH 7.0). All the samples were heated for 5–10 min at 90 °C and slowly cooled (10–12 h) to room temperature. The solutions were equilibrated for several hours at 4 °C. The annealing process was assumed to be complete when the ¹H NMR spectra were superimposable on changing time. NMR spectra were recorded at 25 °C by employing a 700 MHz Bruker spectrometer (Bruker-Biospin, Billerica, MA, USA). Proton chemical shifts were referenced to the residual water signal, resonating at 4.78 ppm (25 °C, pH 7.0). Water suppression was achieved using the excitation sculpting with the gradient routine included in the “zgesgp” pulse sequence [35 (link)]. NMR data processing was done by using the vendor software TOPSPIN 4.1.4 (Bruker Biospin Gmbh, Rheinstetten, Germany).

A 700 MHz Bruker spectrometer was used to perform the NMR experiments. Sample strand concentrations was around 100 µM dissolved in 20 mM potassium phosphate pH 7 and 120 mM KCl in 90% H2O/10% in 5 mm tubes. The 1D-1H-NMR spectra were recorded using a double pulse field gradient perfect spin echo (zgesgppe) pulse sequence to suppress the water signal. Spectra were recorded with a spectral width of 19 ppm, an acquisition time of 1.2s and a relaxation delay of 2s. Experiments were performed at 15°C and 4°C.

The residue obtained after drying was dissolved in 0.6 mL of deuterium oxide with added 100 mM phosphate, keeping the sample at pH 7.2 and 0.1mM TMSP reference. These samples were pipetted into a 5-mm NMR tube for NMR analysis. All ¹H NMR measurements were performed on a Bruker 700 MHz spectrometer at 298 K. 1D ¹H NMR were measured for all samples using 1D NOESY water suppression sequence. 2D JRES pulse sequence follows recommendation made by Parsons et al. [22 (link)]. Due to longer acquisition time 2D JRES spectra were measured only for one representative control sample for each studied wheat variety. For the same samples we also measured 2D TOCSY spectra. 2D JRES and 2D TOCSY spectra were used for assignment of measurable metabolites. All 1D and 2D spectra were processed using MestReNova 9.1.0 software. Spectral preprocessing for 1D spectra included: exponential apodization (exp 1); global phase correction; and normalization using the total spectral area. Spectral regions from 0.5–9.5 ppm were included in the normalization and analysis. 2D spectra were processed using standard procedure recommended in the MestReNova documentation.

All ¹H-NMR experiments were carried out on a Bruker 700 MHz spectrometer (Bruker biospin) equipped with 5 mm TXI probe at 25°C. The jump-and-return water suppression is used in all experiments (35 ). The sweep widths were 20 ppm with a 3-sec relaxation delay with a size of 32K data points per 1D spectra. The number of scans and dummy scans was 128 and 16 respectively. The 1D raw data were processed and analyzed with Topspin 4.06 software inbuilt with the instrument. All the quadruplex sequences were 100 μM strand concentration in 100 mM TMAA + 1 mM KCl in a 5 mm NMR tube (Wilmad from CortecNet, France).

Although stably folded and monodisperse in solution, s2 with its N-terminal linker did not crystallize. ¹⁵N-enriched apo s2 was produced to measure the dynamics of the protein using NMR. ¹⁵N-enriched s2 was prepared as described in Philominathan et al. (2008 ▶ ). NMR experiments were performed at 298 ± 0.5 K on a Bruker 700 MHz spectrometer equipped with a cryoprobe. The concentration of the protein used was 0.1 mM in 50 mM Tris–HCl pH 7.5. In the HSQC spectra, 13 residues could not be identified owing to line broadening (Supplementary Fig. S1). Using the homology-modeled s2 (based on PDB entry 2c4x; Najmudin et al., 2006 ▶ ), we reasoned that the unobserved HSQC peaks corresponded to a highly dynamic N-terminus that hindered crystallization. Guided by the solution data, 13 residues were truncated from the N-terminus. The truncated s2 crystallized readily.

All NMR spectra (1D ¹H, 2D ¹H–¹⁵N HSQC, and 2D ¹H–¹⁵N TROSY HSQC) were acquired at 25°C on a Bruker 700 MHz spectrometer equipped with a cryogenic probe or a room temperature probe. Uniformly ¹⁵N-labelled protein samples were prepared in the NMR buffer containing 20 mM Tris–HCl pH 7.4 at 25°C, 50 mM NaCl, 2 mM DTT, 0.02% NaN₃ for most of the titration experiments. The buffer used for NMR titration experiments of trimeric RPA with HHD1+2, HHD2 mutants with 32C, and ssDNA-22 with HHD2 at pH 6.5 is mentioned in the protein purification section and/or result section. 10% D₂O (v/v) (Cambridge Isotope Laboratories; Cat. No. DLM-4–25) was added to the sample for the spectrometer deuterium lock. Typically, for the titration experiments, protein sample concentration was kept at 150 μM (in case of cryoprobe) or 250 μM (room temperature probe).
The NMR data were processed using Bruker Topspin and analysed using the NMRFAM-SPARKY software (43 (link)).
The chemical shift perturbations (CSPs) were analysed as combined amide chemical shift changes with equation: Δδ_NH (ppm) = [(Δδ¹H)² + (Δδ¹⁵N/5)²]^1/2, where the chemical shift changes in the ¹H and ¹⁵N dimensions are denoted by Δδ¹H and Δδ¹⁵N respectively.