Computational protocol: Partial thioamide scan on the lipopeptaibiotic trichogin GA IV. Effects on folding and bioactivity

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[…] All 1H NMR experiments were performed on a Bruker AVANCE DMX-600, DRX-400 or AC200 spectrometer operating at 600, 400 or 200 MHz, respectively, using the TOPSPIN software package. Splitting patterns are abbreviated as (s) singlet, (d) doublet, (t) triplet, (q) quartet, (m) multiplet. The homonuclear 2D spectra of the three monothionated [Leu11-OMe] trichogin GA IV analogues were recorded at 298 K, with CD3CN as solvent. All spectra were acquired by recording 512 experiments, each one consisting of 64–80 scans and 2000 data points. The spin systems of protein amino acid residues were identified by using standard DQF-COSY [] and CLEAN-TOCSY [–] spectra. In the latter case, the spin-lock pulse sequence was 70 ms long. A ROESY experiment was exploited for sequence-specific assignment. The mixing time of the ROESY experiment acquired for ψ[CS-NH] 9 (peptide concentration: 1.00 mM in CD3CN) and used for interproton distance determination was 200 ms. Interproton distances were obtained by integration of the ROESY spectra using SPARKY 3.111. The calibration was based on the average of the integration values of the cross peaks due to the interactions between the sequential amide protons, set to a distance of 2.80 Å. When peaks could not be integrated because of partial overlap, a distance corresponding to the maximum limit of detection of the experiment (4.0 Å) was assigned to the corresponding proton pair.MD calculations were carried out using the SA protocol of the XPLOR-NIH 2.9.6 program []. For distances involving equivalent or non-stereo-assigned protons, an r−6 averaging was used. The MD calculations involved a minimization stage of 100 cycles, followed by SA and refinement stages. The SA consisted of 30 ps of dynamics at 1,500 K (10,000 cycles, in 3 fs steps) and of 30 ps of cooling from 1,500 to 100 K in 50 K decrements (15,000 cycles, in 2 fs steps). The SA procedure, in which the weights of ROE and nonbonded terms were gradually increased, was followed by 200 cycles of energy minimization. In the SA refinement stage, the system was cooled from 1,000 to 100 K in 50 K decrements (20,000 cycles, in 1 fs steps). Finally, the calculations were completed with 200 cycles of energy minimization by using a NOE force constant of 50 kcal/mol. The generated structures were visualized with the MOLMOL [] (version 2K.2) program. […]

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