Computational protocol: Structural and Functional Characterization of a Novel α-Conotoxin Mr1.7 from Conus marmoreus Targeting Neuronal nAChR α3β2, α9α10 and α6/α3β2β3 Subtypes

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Protocol publication

[…] Samples of CTX Mr1.7 were prepared by dissolving peptides into 500 μL of either 9:1 (v/v) H2O/D2O or 99.99% D2O (Cambridge Isotope Lab, Andover, MA, USA) containing 0.01% TFA at pH 3.0. The final peptide concentration was approximately 3.0 mM. NMR spectra were collected on Bruker Avance 400 and 600 MHz NMR spectrometers at 298 K. The homonuclear DQF-COSY, TOCSY and NOESY spectra were obtained in a phase-sensitive mode using time-proportional phase incrementation for quadrature detection in the t1 dimension. Presaturation during the relaxation delay period was used to suppress the solvent resonance, unless specified otherwise. NOESY spectra were obtained with a mixing time of 300 ms. TOCSY spectra were collected using the MLEV-17 pulse scheme for a spin lock of 120 ms. In order to identify the slow exchange of backbone amide protons, each sample lyophilized from the hydrogen-containing solution was re-dissolved in a deuterium-containing solution. All chemical shifts were referenced to the methyl resonance of 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) used as internal standard. The spectra were processed using Bruker Topspin 2.1 and analyzed by Sparky 3.1 [].Structural calculations were performed with distance constraints derived from the NOESY spectra of Mr1.7 using CYANA 2.1 software []. Dihedral angle restraints were determined based on the 3JHN-Ha coupling constants derived from the DQF-COSY spectral analysis. The φ angle constraints for some residues were set to −120 ± 40° for 3JHN-Ha > 8.0 Hz and −65 ± 25° for 3JHN-Ha < 5.5 Hz, respectively. In addition, backbone dihedral constraints were not applied for 3JHN-Ha values ranging from 5.5 Hz to 8.0 Hz. Based on the slow exchange of amide protons in hydrogen-deuterium exchange experiments, the hydrogen bond constraints were added as target values of 2.2 Å and 3.2 Å for the NH(i)–O(j) and N(i)–O(j) bonds, respectively. The 20 lowest energy conformers were submitted to a molecular dynamics refinement procedure using the Sander module of the Amber 9 program. The final outcomes were used for structural quality analysis using MOLMOL software, and the geometric qualities of the refined structures were evaluated using PROCHECK-NMR software. The data, including chemical shifts, were submitted to the BMRB database with access code 19639 for Mr1.7. […]

Pipeline specifications

Software tools Sparky, CYANA, MOLMOL, PROCHECK
Databases BMRB
Applications NMR-based proteomics analysis, Protein structure analysis
Chemicals Acetylcholine