Drx 800 mhz spectrometer

BL21-DE3 Escherichia coli cells expressing N-terminally His-tagged, GB1-RevARM or GB1-RevDO were grown in M9 minimal medium supplemented with 4 g of glucose as the carbon source and 1 g of ¹⁵N NH4Cl for uniform ¹⁵N labeling. Protein expression and purification were performed as described for the unlabeled protein.
Tag-cleaved Rev was mixed with RNA (IA or IIB) at a 1.2:1 (RNA/protein) ratio, concentrated, and purified on a Superdex 75 size exclusion column equilibrated in NMR buffer (25 mM HEPES pH 6.5, 0.1 M KCl, 1 mM MgCl₂, 0.5 mM EDTA). Fractions containing the Rev/RNA complex were pooled and concentrated to 0.4 mM.
¹H–¹⁵N HSQC NMR spectra^{27 (link)} were acquired at 288 K on a Bruker BioSpin DRX 800 MHz spectrometer equipped with a cryoprobe. Samples contained uniformly labeled ¹⁵N RevDO in complex with IA or IIB at 0.4 mM in NMR buffer with 10% D₂O. Data were processed using NMRPipe^{28 (link)} and analyzed using SPARKY.²⁹ One-dimensional ¹H spectra were acquired using the 1–1 echo pulse sequence³⁰ on samples in NMR buffer with 10% D₂O at 288 K.

Relaxation dispersion experiments report on ms-µs timescale exchange processes. Methyl ¹³C CPMG relaxation dispersion experiments were performed on a [1-¹³C, ¹⁵N] hE:FOL:NADP⁺ sample at multiple temperatures (280 K, 300 K, and 310 K) using a Bruker DRX 800 MHz spectrometer and the pulse schemes described by Skrynnikov et al.^{24 (link)} and Lundström et al.^{25 (link)} Using [1-¹³C] glucose as the sole carbon source leads to isolated ¹³C enriched methyls for most methyl side chains; however, the presence of ¹³C-¹³C_methyl labeling for Thr-γ2 and Ile-δ1 methyls can interfere with accurate spin-relaxation measurements due to the one bond C-C coupling.^{18 (link)} Experiments were performed using a total relaxation period T_CPMG of 40 ms and refocusing delays 1/τ_cp of 100, 200, 400*, 600, 1000, 1400*, 1800, and 1900 s⁻¹ where * denotes experiments completed in duplicate. Dispersion curves were fit using the program GLOVE.^{26 (link)}