Chirascan plus acd spectropolarimeter

The main instruments employed in this study were a Chirascan plus ACD spectropolarimeter (Applied Photophysics, UK), CRC-55tR radiopharmaceutical dose calibrator (Capintec Inc., USA), Wizard 1480 gamma counter (PerkinElmer Instruments Inc., USA), Scan-RAM Radio-HPLC/TLC scanner (LabLogic, UK), and U-SPECT +/CT small-animal SPECT/CT imaging system (MILabs, The Netherlands).

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.

The main equipment used in this study included a Chirascan plus ACD spectropolarimeter (Applied Photophysics, United Kingdom), a CRC-55tR radiopharmaceutical dose calibrator (Capintec Inc., USA), a Wizard 1480 gamma counter (PerkinElmer Instruments Inc., USA), a 20-mCi ⁶⁸Ge/⁶⁸Ga generator (Obninsk Cyclotron Co., Ltd., Russia), and an Inveon small animal PET scanner (Siemens, Germany).

The morphologies of clean samples were observed by a scanning electron microscope (SEM, FEI Quanta 200, 15 kV) after sputter-coating with a thin layer of gold nanoparticles. The Raman spectra were recorded at an excitation wavelength of 514 nm. The spectra were scanned three times for 20 s in the range 100–1400 cm⁻¹ using a LabRAM HR Evolution spectrometer (HORIBA Jobin Yvon). The CLSM images were obtained using a LSM 710 META confocal Microscope (Zeiss), and the excitation wavelength for FITC-labeled samples was set at 488 nm. The X-ray diffraction (XRD) patterns were obtained using a Bruker D8 Advance X-ray diffractometer (Cu Kα, λ = 0.154 nm, 40 kV, 200 mA). The scattered radiation was detected in the angular range 20−80° (2θ) with a scan rate of 1.2°·min⁻¹. Unit cell parameters were calculated by Jade 5. The circular dichroism (CD) measurements were carried out using a Chirascan plus ACD spectropolarimeter (Applied Photophysics) using 5-mm path length cuvettes.

The CD spectra were recorded on a Chirascan-plus ACD spectropolarimeter (Applied Photophysics, Leatherhead, UK) using a 1 mm quartz cuvette at 20 °C. The temperature-scan experiments (20–90 °C) were performed with a 0.5 mm quartz cuvette. For each CD spectrum, four scans were accumulated using a step resolution of 0.1 nm, a bandwidth of 1 nm, a response time of 2 s, and a scan speed of 20 nm/min. The CD spectrum of the solvent was subtracted from that of the protein (Id2’) solution to eliminate interferences from the cell, solvent and optical equipment. The CD signal was normalized as mean-residue ellipticity (deg·cm²·dmol⁻¹) by using the protein concentration obtained by UV measurements on an Agilent Cary 60 UV-Vis spectrophotometer (an extinction coefficient of 1480 M⁻¹·cm⁻¹ per Tyr residue [58 (link)] was applied).