Avance 600 mhz nmr spectrometer

Data used for PaurTx-3 structure calculations was acquired on a Bruker Avance 600 MHz NMR spectrometer (Bruker, Billerica, MA, USA) equipped with a cryoprobe. The solution NMR structure for PaurTx-3 was calculated using previously described protocols [26 (link)], on a Bruker Avance 600 MHz NMR spectrometer equipped with a cryoprobe. CCPNMR Analysis 2.4.1 (CCPN, University of Cambridge, Cambridge, UK) was used for amino acid assignment [46 (link),47 ]. The solution NMR structure for PaurTx-3 was calculated as previously described [26 (link)] using the AUTO and ANNEAL functions in CYANA 3.97 (Güntert Group, Goethe-Univerity Frankfurt, Frankfurt, Germany) [48 (link)] to refine peak assignments. Dihedral angle restraints were generated using TALOS-N (Bax Group, NIH, Pike Bethseda, MD, USA) [49 (link)]. After initial structure determination on CYANA, protocols on the RECOORD database [50 (link)], were used to generate 50 structures which were then refined in a water shell [51 ], and a final set of 20 structures was chosen based on the lowest energy, best MolProbity scores [35 (link)], and fewest distance and dihedral angle violations (Table 1). PaurTx-3 structure and restraints have been submitted to the Protein data bank (PDB ID: 5WE3) and the Biomagnetic Resonance Data bank (BMRB: 30317).

2D ¹H-¹³C heteronuclear single quantum coherence (HSQC) correlation spectra were obtained with uniformly ¹⁵N, ¹³C labeled IL-17A expressed in E. coli (Supplementary Method). Samples containing 50  μM IL-17A and 250 μM compound from linear peptide series (with K_D = 0.1 μM) were prepared in 10 mM HEPES, pH 7.4, 150 mM NaCl containing 10% D₂O. The DMSO concentration was maintained at 3%. NMR data was recorded on a Bruker Avance 600 MHz NMR spectrometer at 298 K. Spectra were acquired using 2048 × 128 complex points and spectral widths of 10,000 × 2300 Hz in t2 × t1 dimensions. The recycle delay was 1 s and 64 transients were recorded for each complex t1 point. All data were processed using TopSpin 2.1 (Bruker Instruments). The F1 dimension was zero filled to 256 points and sine function was applied to both dimensions prior to Fourier transform.
2D nuclear Overhauser effect spectroscopy (NOESY) spectra was recorded for compound 1 at 300 μM in 50 mM Tris-d, 150 mM NaCl in 100% D₂O at pH 7.4 (not corrected for D₂O) on a Bruker Avance 600MHz NMR spectrometer at 288 K and 298 K. Data was collected with 2048 complex points in the F2 dimension and 128 increments in the F1 dimension over a spectral width of 16 ppm and mixing times of 300 ms and 500 ms. The F1 dimension was zero filled to 512 and both dimensions were multiplied by a square – sine function prior to transformation.

The sample was prepared in D₂O at 5 mg/mL of concentration. The ¹H-NMR analysis(Bruker Avance 600 MHz NMR spectrometer, Bruker, Billerica, Massachusetts, USA) was carried out at 600 MHz.