Drx 600 mhz spectrometer

All NMR experiments were performed at 25°C on a Bruker DRX 600 MHz spectrometer. NMR samples were prepared with 90% of NMR buffer (20 mM HEPES pH 7.5 and 150 mM NaCl) and 10% of D₂O. NMR titrations were carried out by acquiring ¹H- ¹⁵N heteronuclear single quantum correlation (HSQC) spectra of 0.2 mM of ¹⁵N-labeled protein. Subsequent spectra were taken after the addition of an unlabeled ligand. ¹⁵N-labeled CMG P-domain (residues 300–369) was titrated with unlabeled PDILT b′ domain to the final ratio of labeled to unlabeled protein of 1:2. Purified mastoparan (INLKALAALAKKIL) peptide was added to the ¹⁵N-labeled PDILT b′ domain to a final protein/peptide ratio of 1:10. Also, ¹⁵N-labeled PDILT b′ domain was titrated with unlabeled CRT3 P-domain (residues 198-291) to a ratio of labeled/unlabeled protein of 1:2. Specta were processed by NMRPipe33 (link) and analyzed by NMRview.

The water NMR samples were prepared in 25 mM K-phosphate and 70 mM KCl buffer at pH 7 in D₂O/H₂O (10%/90%). The D₂O samples were lyophilized and redissolved in 99.98% D₂O two more times. Each sample was heated to 95 °C for 5 min and cooled slowly to room temperature. The final concentrations of DNA oligonucleotides were 0.1–2.5 mM. NMR experiments were performed on a Bruker DRX-600 MHz spectrometer Standard homonuclear 2D NMR experiments, including DQF-COSY, TOCSY and NOESY, were collected at temperatures of 5, 15, 25 and 35 °C for the BMVC and MycG4 complex samples in water and D₂O solution in 95 mM K⁺ at pH 7. The mixing times were set from 50–250 ms for NOESY, and at 40 ms and 80 ms for TOCSY experiments. The NMR experiments for samples in water solution were performed with WATERGATE or jump-return (NOE11) water suppression techniques. Peak assignment and integration were achieved using Sparky (UCSF). Distances between protons were assigned based on the nuclear overhauser effect (NOE) cross peaks integrated at 200 ms mixing times, with the upper and lower boundaries assigned to ±20% of the estimated distances. The methyl base proton Me-H6 distance (2.99 Å) was used as a reference. The distances involving the unresolved protons, e.g. methyl protons, were assigned using pseudo-atom notation in X-PLOR.

For each pH value (4.0 and 7.0), a 2D [¹⁵N,¹H] sfHSQC spectrum (ns: 8, TD_1H: 2k, TD_15N: 128) of 0.4 mM sample (U-¹⁵N) in NMR buffer was recorded. All spectra were acquired on a Bruker DRX 600 MHz spectrometer at 298 K. All data were processed by TopSpin 3.5 and spectra were analysed using CCPNMR 2.4.2 (53–55 (link)).