Computational protocol: Designed multi-stranded heme binding β-sheet peptides in membrane††Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc04108b

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Protocol publication

[…] All NMR experiments were performed at 42 °C on a Bruker DRX 600-MHz spectrometer equipped with a cryo-probe. NMR samples were prepared by dissolving the lyophilized peptide in 500 μL 90% H2O/10% D2O containing 125 mM per-deuterated DPC. The concentration of the NMR sample was approximately 0.3–0.5 mM. Two dimensional TOCSY (mixing time, 80 ms) and NOESY (mixing time, 200 ms) experiments were acquired for spin system assignments and structure determination. The chemical shifts of protons were internally referenced to 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS). The proton resonances of all the amino acids were assigned unambiguously by overlaying TOCSY and NOESY spectra and sequential walking. Backbone dihedral angles were then predicted after obtaining Hα, an amide proton, (and 13Cα chemical shifts) of the assigned NOESY and 13C–1H HSQC spectra using PREDATOR. NOE restraints were derived from the NOE intensities of the assigned NOESY spectra and categorized into strong, medium and weak, respectively. This was further translated to distance limits between 2.5 and 5.0 Å. The NOE restraints and predicted dihedral angle values were used to carry out several rounds of structure calculations using CYANA 2.1. Of the 100 structures generated, 20 energy minimized structures were selected for evaluation and analysis. PROCHECK-NMR was used to assess the stereo-chemical quality of the structure ensembles. The structures were analyzed visually by PYMOL and MOLMOL. PRE experiments were carried out by titrating 2 mM 16-doxyl-stearic acid (16-DSA) dissolved in deuterated methanol to a lyophilized peptide sample in DPC micelles. Two dimensional TOCSY spectra were carried out with and without the PRE probe using the same experimental conditions. The intensities of the CαH/NH cross peaks were evaluated before and after the addition of the PRE probe and their ratios plotted for each amino acid. To facilitate MTSL (S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate) labelling, peptide-7H6C was incubated with 10 : 1 MTSL : peptide concentration in 50% acetonitrile solution and incubated overnight at room temperature (>12 hours). In-order to remove excess MTSL and unlabelled peptide, the incubated sample was diluted and subjected to HPLC purification. The major eluted peaks were analyzed by mass spectrometry and the labeled peptide fraction was verified. The labeled peptide was then reconstituted in identical buffer conditions and two dimensional TOCSY spectra were carried out for the labelled and unlabelled peptide. The intensity of the TOCSY peaks observed for the labelled sample were used as a measure to derive distance restraints for the peptide-7 structure. […]

Pipeline specifications

Software tools CYANA, PROCHECK, PyMOL, MOLMOL
Applications NMR-based proteomics analysis, Protein structure analysis
Chemicals Amino Acids, Heme