J1700 cd spectrometer

Circular dichroism (CD) was applied evaluate secondary structures in protein in the far UV range (190–260 nm) and to monitor protein-bound co-factors such as iron-sulfur clusters in the near UV-Visible range (300–600 nm) in this study. As for the protein-bound iron-sulfur cluster types analysis, purified wild-type MagR protein (clMagR^WT) and mutants were prepared at 100 μM in TBS buffer (20 mM Tris, 150 mM NaCl, pH 8.0) and measured in 1 cm diameter quartz cells at room temperature using a MOS-500 (Biologic) CD Spectrometer. To analyze the secondary structure and thermal stability of wild type MagR and its mutants, measurements were performed using a J-1700 CD Spectrometer (JASCO Corporation, JPN) in the Far-UV range (190–260 nm). Purified wild-type MagR protein (clMagR^WT) and mutants were prepared at 10 μM protein (20 mM Na₂HPO₄, pH 8.0) in 1 mm quartz cuvettes at room temperature. The thermal stability of wild-type MagR protein (clMagR^WT) and mutants were monitored and recorded by CD spectrum in the range of 25°C to 95°C with temperature increases at 1°C intervals. The melting temperature values were calculated by sigmoidal fitting of the thermal denaturation curve at 222 nm using Boltzmann function.

All solvents were commercially available and utilized without further purification. All unmodified DNA sequences used in this study were ordered from Sangon Biotech. The DNA phosphoramidites and CPG were purchased from DNA Chem. The HEK293T cell line was obtained from SUNNCELL (catalog number: SNL-015) and used without authentication testing. The compound masses were weighed on a microbalance with a resolution of 0.1 mg. TLC (Thin-layer chromatography) analysis was performed through pre-coated silica gel plates. Column chromatography carried out by silica gel (#100–200). ¹H, ¹³C and ³¹P NMR spectra for compound characterizations (Supplementary Fig. 21–44) were recorded on Bruker AVANCE III 400 MHz spectrometer. The mass spectra (MS) were recorded on LCMS-2010A. UV absorbance was recorded by the Shimadzu 2600 UV-Vis spectrophotometer. Circular dichroism spectra were recorded by the Jasco J1700 CD Spectrometer. ITC experiments were carried on MicroCal iTC200 at 298 K. Gel shift was imaged by Gel Image System (Tanon 2500 R). The fluorescence was recorded by a SHIMADZU-RF-6000 fluorescence spectrophotometer. Flow cytometry was measured by the BD FACSCanto II flow cytometry instrument (BD Biosciences).