Spectra analysis

Measurements were carried out using an FTIR spectrometer (Jasco, 6800 FV, Tokyo, Japan) equipped with a Diamond ATR device (Jasco, ATR Pro One, Tokyo, Japan). The effective dimensions of the diamond are 1.8 mm diameter. The refractive index of the diamond is 2.4 and the angle of incidence in our device was 45°, generating 1 reflection. The calculated depth of penetration is ~ 2 μm. The radiation from the IR source of the spectrometer was focused into the ATR diamond, and the output radiation (from the other side of the diamond) was focused onto a DLaTGS (Deuterated Lanthanum α Alanine doped TriGlycine Sulphate) detector. 10–20 µl of cell suspensions of specific lineages were placed on the diamond ATR. Pressure (700 kg/cm²) was applied to produce better contact between the sample and the diamond. Measurements were carried out in the spectral range of 4000–650 cm⁻¹. Each spectrum at acquisition (see Supplementary) was an average of 64 scans to increase the signal to noise ratio (SNR). The spectra were analyzed using Spectra Analysis™ (Jasco, Tokyo, Japan).

Circular dichroism of proteins (0.1 mg/ml in CD buffer: 10 mM Na phosphate buffer, pH 8.0) as a function of wavelength (190–350 nm, 1 nm interval) was measured using a J-720 machine (Jasco). Five independent measurements were averaged and smoothed using Means-Movement with a convolution width of 11 (Spectra Analysis, JASCO). The spectrum of the buffer solution was subtracted prior to the deconvolution of spectra using the Dichroweb server [32 (link)] employing the CDSSTR program and reference set 4.

All Circular Dichroism (CD) experiments were performed using a JASCO J-1500 Spectropolarimeter (JASCO Inc., Tokyo, Japan) using a 0.1-cm path length cell. All samples were exchanged into 25 mM potassium phosphate (pH 7.0). Analysis of CD spectra was performed using Spectra Analysis (JASCO Inc., Tokyo, Japan). To test the effect of hydrogen peroxide on global protein structure of CsOxOx, spectra were recorded of a 1.0 mg/mL solution at 4 mM, 8 mM, 12 mM, 16 mM, and 20 mM hydrogen peroxide at 25°C. In order to observe the effects of thermal denaturation on the secondary structural elements of CsOxOx, spectra were recorded of a 0.68 mg/mL solution at increasing temperatures (10°C to 90°C, with 10°C increments). Each spectrum was taken after ten minutes of incubation at each temperature. A Teflon stopper was used to retard evaporation. The scan rate, time constant and numbers of scans were 10 nm/min, 2 s, and 3, respectively. Each spectrum was an accumulation of five scans. A blank spectrum was performed with buffer and subtracted from the spectra.