NMR spectroscopy and structure calculations

The NMR sample was prepared by dissolving guavanin 2 in a micellar solution containing 100 mM of deuterated dodecylphosphocholine (DPC-d₃₈) and 5% D₂O at 1 mM concentration. The pH was adjusted to 4.0. All spectra were acquired at 25 °C on a Bruker Avance III 500 spectrometer equipped with a 5 mm triple resonance broadband inverse (TBI) probehead. Proton chemical shifts were referenced to sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) and water suppression was achieved using the pre-saturation technique. ¹H-¹H TOCSY experiment was recorded with 128 transients of 4096 data points, 256 t1 increments, and a spinlock mixing time of 80 ms. The ¹H-¹H NOESY was recorded with 64 transients of 4096 data points, 256 t1 increments, mixing time of 250 ms. Spectral width of 8012 Hz in both dimensions. ¹H-¹³C HSQC experiment was acquired with F1 and F2 spectral widths of 8012 and 25152 Hz, respectively, were collected 256 t1 increments with 96 transients of 4096 points for each free induction decay. The experiment was acquired in an edited mode. All NMR data were processed using NMRPIPE⁶¹ and analyzed with NMR View⁶².

The structure calculations were performed with the XPLOR-NIH version 2.28 software by simulated annealing (SA) algorithm⁶³. NOE intensities were converted into semi-quantitative distance restrains using the calibration by Hyberts et al.⁶⁴. The angle restraints of phi and psi of the protein backbone dihedral angles were predicted based on analysis of ¹H_α and ¹³C_α chemical shifts using the program TALOS+⁶⁵. Several cycles of XPLOR were performed using standard protocols. After each cycle rejected restraints, side-chain assignments, NOEs, and dihedral violations were analyzed. Two hundred structures were calculated, and among them, the 20 lowest energy structures were submitted to XPLOR-NIH water refinement protocol⁶³. The ensemble of the 10 lowest energy conformations was chosen to represent the solution structure ensemble of guavanin 2.

The restrictions used in structural calculations were analyzed by QUEEN program (Quantitative Evaluation of Experimental NMR Restraints). This program performs a quantitative assessment of the restrictions of the experimental NMR data. QUEEN checks and corrects possible assignments of errors by the analysis of the restrictions⁶⁶. The stereochemical quality of the lowest energy structures was analyzed by PROCHECK⁵⁰ and ProSA⁴⁹. PROCHECK was used in order to check stereochemical quality of protein structure through the Ramachandran plot, where good quality models are expected to have more than 90% of amino acid residues in most favored and additional allowed regions. ProSA indicates the fold quality by means of the Z-score. The display, analysis, and manipulation of the three-dimensional structures were performed with the program MOLMOL⁶⁷ and PyMOL (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC).