Vnmrs 600 and 800 mhz spectrometer

All NMR experiments were conducted using 400 μM ¹⁵N, ¹³C-RNase P protein in 20 mM sodium phosphate pH 6.0, 80 mM NaCl, 50 μM 4, 4-dimethyl-4-silapentane-sulfonate (DSS) at 318 K with 5 % (v/v) D₂O in a 3 mm Norell® select series NMR tube unless noted. NMR spectra were acquired on Bruker Avance-III 600MHz, 800MHz and 900 MHz spectrometers, equipped with cryogenic probes, as well as Varian VNMRS 600 and 800 MHz spectrometer equipped with a cryogenic probe. For backbone assignments, (¹H, ¹⁵N) heteronuclear single quantum coherence (HSQC) and a set of traditional triple-resonance experiments were performed, including HNCO, HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, HN(CA)CO. Side-chain assignments were carried out using three-dimensional (H)C(CCO)NH, H(CCCO)NH, (H)CCH-TOCSY, H(C)CH-TOCSY, ¹⁵N-NOESY-HSQC, ¹³C-NOESY-HSQC and (¹H, ¹³C) HSQC for aliphatic and aromatic groups, respectively. The ¹³C-NOESY-HSQC and aliphatic and aromatic optimized 2D ¹³C HSQC experiments were obtained in 95% (v/v) D₂O. ¹H chemical shifts were referenced with respect to internal DSS, and ¹³C and ¹⁵N chemical shifts were referenced indirectly using nuclei-specific gyromagnetic ratios (Wishart et al. 1995 (link)). Spectra were processed with Topspin 3.5.7 (Bruker Inc.) and NMRPipe (Delaglio et al. 1995 (link)), and analyzed by using CARA (Keller 2004 ) and Sparky (Goddard and Kneller).