Topspin v1

All NMR spectra were recorded on a DRX 500-MHz NMR spectrometer (Bruker, Billerica, MA, USA) using a 5-mm BBO probe with a z-gradient. Hydroxyprotoilludene samples were dissolved in deuterated dichloromethane to a final concentration of ~ 50 mM and were calibrated against the solvent signals at 5.32 and 54.00 ppm. ¹³C-NMR spectra was acquired by conducting 1000 scans with a 30° pulse and a delay of 2 s. ¹³C-DEPT-135 spectra was also acquired by conducting 400 scans. All 2D spectra were acquired using Bruker standard pulse programs. The DQF-COSY and gs-NOESY spectra were measured in 512 increments, with two or four scans per increment, respectively. A mixing time of 600 ms was used for the acquisition of the NOESY spectrum. For heteronuclear correlation, the gs-HSQC and gs-HMBC spectra were measured in 512 increments, with four or eight scans per increment, respectively. The spectra were optimized for ¹J and ⁿJ couplings of 140 and 8 Hz, respectively. The data were processed using TopSpin v1.3 (Bruker) and MestReNova v8.0 (MestreLab Research, Santiago de Compostela, Spain).

NMR experiments were performed at 25°C using Bruker Avance 600-MHz NMR spectrometers equipped with a 5-mm single-axis z gradient cryoprobe. Spectra were recorded using band-selective, optimized flip angle short transient, ¹H, ¹⁵N-heteronuclear multiple-quantum coherence (SOFAST-HMQC) [29 (link)]. 1024 data points were acquired in the direct ¹H dimension and 96 points in the indirect ¹⁵N dimension, with a recycle delay of 200 ms. ¹⁵N-enriched RPA70N was prepared at 26 μM in buffer containing 25 mM Tris-Cl at pH 7.5, 100 mM NaCl and 1 mM DTT. A series of ¹⁵N-¹H HSQC spectra were collected at RPA70N/Tag(84–130) ratios of 1:0, 1:0.5. 1:1, 1:2, 1:4, 1:8, 1:12 and 1:16. All spectra were processed with TOPSPIN v1.3 (Bruker, Billerica, MA) and analyzed with Sparky v3.1 (University of California, San Francisco, CA). ¹H and ¹⁵N backbone NMR assignments for RPA70N were reported previously [30 (link)]. Chemical shift perturbations (Δδ) were calculated using a weighted average of net changes in chemical shift in both ¹H and ¹⁵N induced by binding of Tag(84–130) using following equation: