Brevinin-1GHd Peptide Structural Analysis

Circular dichroism (CD) spectroscopy of Brevinin-1GHd was subjected using Chirascan plus ACD spectropolarimeter (Applied Photophysics Ltd., Leatherhead, United Kingdom) as previously described by us to estimate its secondary structure and interaction with LPS (L2880, Escherichia coli O55: B5, Sigma-Aldrich, St. Louis, Missouri, MO) (Wu et al., 2021 (link)). Shortly, Brevinin-1GHd at the final concentration of 50 μM was dissolved in SDS solution (0, 30, 60, 90, and 120 mM) or in 60 mM SDS solution before incubation at different temperatures (25, 37, 50, 70, and 90°C) for 1 h, and the CD spectrum was subsequently determined. To explore the binding of Brevinin-1GHd to LPS, 0.2 mg/ml of LPS was dissolved in H₂O or 30 mM SDS solution, and then was mixed with 50 μM Brevinin-1GHd for 1 h at 25°C. Soon afterwards, the CD spectrum was determined. The binding of Brevinin-1GHd with LPS was identified by comparing the changes of spectrogram of each sample. CD data are presented as the mean residue ellipticity of three consecutive scans per sample in θ, deg.·cm²·dmol⁻¹. The mean residue ellipticity (θ, deg.·cm²·dmol⁻¹) is calculated by following equation: $\theta ={\theta }_{obs}/\left(10\times c\times l\times n\right)$ , where θ_obs is the observed ellipticity (mdeg), c is the concentration (mol/L) of Brevinin-1GHd solution, l is the path length (cm), and n is the number of Brevinin-1GHd residues.