500 mhz and 800 mhz nmr spectrometers

NMR samples were prepared in 15 mM sodium acetate and pH 5.0. The protein was quantified by UV-Vis spectrophotometry using a theoretical extinction coefficient of 7082 M⁻¹cm⁻¹. All NMR experiments were recorded at 298 K on samples containing 0.5–0.7 mM ¹H/¹³C/¹⁵N-labeled RNase 4 in 15 mM sodium acetate at pH 5.0 with 10% ²H₂O in 5-mm Shigemi NMR tubes filled with 350 μL of protein solution. NMR experiments were carried out on Agilent 500 MHz and 800 MHz NMR spectrometers equipped with triple-resonance cold probes and pulsed-field gradients. For backbone resonance assignments of wild-type human RNase 4, 2D HSQC, 3D TOCSY-HSQC and 3D NOESY-HSQC were collected and sequence-specific assignments of the backbone atoms were achieved by running a series of independent CBCA(CO)NH, HNCACB and HNCO 3D experiments. All heteronuclear NMR experiments were processed by NMRPipe and analyzed using Sparky and NMRView.

All NMR experiments were carried out on Agilent 500 MHz and 800 MHz NMR spectrometers equipped with triple-resonance cold probes and pulsed-field gradients. NMR spectra were recorded at 298 K on samples containing 0.75 mM ¹⁵N-labeled RNase A (or the A109G variant) in 5 mM MES-NaOH buffer (pH 6.4) with 7 mM NaCl, 0.01% NaN₃, and 10% ²H₂O. Backbone resonances were assigned by standard multidimensional experiments (Cavanagh et al., 2007 ). Relaxation-compensated ¹⁵N-CPMG experiments were acquired in an interleaved fashion with T_cp¹⁵N-CPMG repetition delays of 0.625, 0.714 (×2), 1.0, 1.25, 1.67, 2.0, 2.50 (×2), 3.33, 5.0, and 10 ms, using a total relaxation period of 40 ms. All NMR spectra were processed using NMRPipe (Delaglio et al., 1995 (link)), in-house scripts and analyzed with Sparky (Goddard and Kneller ). Global residue fits and model analyses were performed by fitting 500 MHz and 800 MHz relaxation dispersion data to the full single-quantum ¹⁵N-CPMG equation (Manley and Loria, 2011 ) using GraphPad Prism 5. NMR titration experiments were performed as previously described (Gagne et al., 2012 (link)), using the single-nucleotide ligands 3′-UMP and 5′-AMP reconstituted in the NMR buffer.