Timecontrol software

NanoDSF was performed using Prometheus NT.48 equipped with backreflection mode (NanoTemper Technologies, München, Germany). Samples were loaded in nanoDSF grade standard capillaries (NanoTemper Technologies GmbH, München, Germany) and exposed at thermal stress from 20 °C to 95 °C by thermal ramping rate of 1 °C/min. Fluorescence emission from tryptophan after UV excitation at 280 nm was collected at 330 nm and 350 nm with dual-UV detector. Protein aggregation was assessed simultaneously employing backreflection optics, which detects protein aggregation by measuring the attenuation of backreflected light intensity passing through the sample. Thermal stability parameters, including T_onset, T_m, and T_agg, were calculated by PR.ThermControl software (NanoTemper Technologies, München, Germany). For isothermal stability, the time interval data from thermal stress at constant temperature (50, 54, 57, or 60 °C) were collected by PR.TimeControl software (NanoTemper Technologies, München, Germany).

The temperature-dependent unfolding and the thermostability of LPORs were determined using differential scanning fluorimetry (DSF), which detects the unfolding of proteins by monitoring the temperature-dependent changes in the fluorescence of the aromatic amino acids of proteins. For Nano-DSF measurements, a Prometheus NT.Flex (NanoTemper Technologies GmbH, Munich, Germany) instrument was used. Purified LPOR samples (10 µl) with a concentration of 0.6 mg ml⁻¹ were subjected to a linear unfolding ramp (0.5 °C min⁻¹, from 15 to 85 °C). The intrinsic tryptophan fluorescence of the protein was monitored continuously (18 data points per minute) at 350 and 330 nm. Unfolding transition midpoints were determined from the first derivative of the fluorescence ratio (F350/F330) by using the RT.ThermControl Software (NanoTemper Technologies GmbH). Thermostabilities were measured at different temperatures for 30 h by using the RT.TimeControl Software (NanoTemper Technologies GmbH).