Computational protocol: A novel lipid transfer protein from the pea Pisum sativum: isolation, recombinant expression, solution structure, antifungal activity, lipid binding, and allergenic properties

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[…] NMR investigation was done using 1.0 mM sample of the 13C,15N -labeled Ps-LTP1 in 5 % D2O at pH 5.5 and 30 °C. NMR spectra were acquired on Bruker Avance 700 spectrometer equipped with a room-temperature triple-resonance (1H, 13C, and 15N) probe. Backbone resonance assignment was obtained using the standard set of 3D triple-resonance experiments []. 3D 13C-HCCH-TOCSY and 15N- or 13C-filtered 3D TOCSY and NOESY spectra were used for side chains assignment. The 3JHNHα and 3JNHβ coupling constants were measured using 3D HNHA and HNHB experiments []. 3JCγC’, 3JCγN constants for Val, Ile, and Thr residues were quantitatively calculated from the cross-peak intensities in the spin-echo difference 13С-HSQC experiments []. Temperature coefficients of amide protons (Δδ1HN/ΔT) were measured over a temperature range 20–70 °C using 2D 15N-HSQC spectra. Heteronuclear 15N-{1H} NOE values were measured at 30 °C using pseudo 3D experiment [].Spatial structure calculation was performed in the CYANA 3.0 program []. Upper interproton distance constraints were derived from the intensities of cross-peaks in 3D 15N-NOESY-HSQC and 13С-NOESY-HSQC (τm = 80 ms) spectra via a “1/r6” calibration. 1H, 13C, and 15N backbone chemical shifts were used as an input for the TALOS+ software to predict the secondary structure []. Torsion angle restraints and stereospecific assignments were obtained from J coupling constants, NOE intensities and TALOS+ predictions. Hydrogen bonds were introduced basing on Δδ1HN/ΔT values. The disulfide bond connectivity pattern was established on the basis of the observed NOE contacts and verified during preliminary stages of the spatial structure calculation. The location and volume of the cavities in the proteins were calculated using CASTp with a 1.4 Å probe radius ( []. The atomic coordinates for the major structural form of Ps-LTP1 have been deposited in the PDB under accession code 2N81. […]

Pipeline specifications

Software tools CYANA, TALOS+, CASTp
Application NMR-based proteomics analysis
Organisms Pisum sativum, Homo sapiens
Chemicals Amino Acids