Computational protocol: Synergistic Activity of the Plant Defensin HsAFP1 and Caspofungin against Candida albicans Biofilms and Planktonic Cultures

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Protocol publication

[…] Dry powder (1 mg) of rHsAFP1 was dissolved in 500 μL of 10% D2O/90% H2O (~pH 4) for NMR experiments. Spectra were recorded at 298 K on a Bruker Avance-600 spectrometer. Two-dimensional NMR experiments included total correlation spectroscopy (TOCSY []) using a MLEV-17 spin lock sequence [] with a mixing time of 80 ms; nuclear Overhauser effect spectroscopy (NOESY []) with a mixing time of 150, 200, or 300 ms; exclusive correlation spectroscopy (ECOSY []); and 13C and 15N heteronuclear single-quantum coherence (HSQC []). Solvent suppression was achieved using excitation sculpting with gradients []. Spectra were acquired with 4096 complex data points in F2 and 512 increments in the F1 dimension. Slowly exchanging amide protons were identified by spectra also recorded in 100% D2O.Spectra were processed using TopSpin (Bruker) software. The t1 dimension was zero-filled to 1024 real data points, and 90° phase-shifted sine bell window functions were applied prior to Fourier transformation. Chemical shifts were referenced to internal 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). Processed spectra were analysed and assigned using CcpNmr Analysis []. Spectra were assigned using the sequential assignment protocol []. [...] Structure calculations were based on distance restraints derived from NOESY spectra recorded in both 10% and 100% D2O. Initial structures were generated using the program CYANA [], followed by addition of restraints for the disulfide bonds, hydrogen bonds as indicated by slow D2O exchange and sensitivity of amide proton chemical shift to temperature, chi1 restraints from ECOSY and NOESY data, and backbone phi and psi dihedral angles restraints generated using the program TALOS+ []. The structural family was generated using torsion angle dynamics, refinement and energy minimization in explicit solvent and protocols as developed for the RECOORD database [] within the program CNS []. A family of structures consistent with the experimental restraints was then visualized using MOLMOL [] and assessed for stereochemical quality using MolProbity []. Coordinates and NMR chemical shift assignments have been submitted (PDB ID: 2n2q; BMRB ID: 25605). […]

Pipeline specifications

Software tools CcpNmr, TALOS+, CNS, MOLMOL, MolProbity
Databases BMRB RECOORD
Applications NMR-based proteomics analysis, Protein structure analysis
Organisms Candida albicans, Saccharomyces cerevisiae, Komagataella pastoris, Fusarium culmorum, Homo sapiens
Chemicals Amphotericin B, Cysteine