Luxeon lumileds

Steady-state UV/Vis spectrophotometric measurements were carried out under dim-red safety light using a Cary-60 UV/Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) equipped with a peltier-thermostatted (25 ± 2 °C) single-cell cuvette holder. All samples were kept in the dark for at least 2 hours before use. All iLOV-Q489D samples were diluted (1:10) to an OD_450nm of 0.1 in 200 mM sodium phosphate buffer pH 7.2 supplemented with 10 mM NaCl. For the measurement under aerobic conditions sample illumination was achieved by using a blue-light emitting high-power LED (Luxeon Lumileds, Phillips, Aachen, Germany) (0.13 mW cm⁻²) mounted on top of the cuvette. LED illumination was controlled using an Arduino UNO (Smart Projects, Italy) microcontroller as described previously^{66 (link)}. Illumination was pulsed (5 seconds illumination time) to precede each sequential UV/Vis scan and spectra were recorded every 30 seconds for up to 15 minutes. To monitor photoreduction in the absence of oxygen, the respective diluted protein solution was degassed by bubbling the solution for 15 minutes with Argon in a 1 cm quartz cuvette closed with a rubber septum. The corresponding degassed protein solution was illuminated for up to 45 minutes by removing the cuvette from the sample holder and illuminating the solution from the side of the cuvette.

Spectroscopic measurements were carried out with a Shimadzu UV-1800 UV-Vis spectrometer (Shimadzu, Kyoto, Japan). For generation of the light state, the protein (in buffer 10 mM NaH₂PO₄/Na₂HPO₄, 10 mM NaCl pH 8.0) was illuminated for at least 30 s using a 450 nm blue-light LED with a radiant power of 50 mW (Luxeon Lumileds, Phillips, Aachen, Germany). Dark recovery kinetics was measured from the illuminated sample by recording the absorption recovery at 475 nm, as described previously30 (link).

All spectroscopic analyses were carried out under red light at 20°C, using either a Beckmann UV/Vis spectrophotometer DU-650 for absorbance and recovery kinetic measurements or by employing a Perkin-Elmer Luminescence Spectrometer LS 50B for fluorescence measurements as described previously [24 (link)]. For all measurements, the samples were diluted to an OD₄₅₀ of 0.1 with 10 mM phosphate buffer, pH 8.0 supplemented with 300 mM NaCl. In order to generate the LOV proteins light state, the samples were illuminated for 30 s using a high-power blue-light (450 nm)-emitting LED (Luxeon Lumileds, Phillips, Aachen Germany). Dark state recovery was measured from illuminated samples by recording the absorption recovery at 485 nm. Reported adduct state lifetimes are derived from three independent measurements and were obtained by fitting experimental data to a mono-exponential decay function using software Origin (OriginLab corporation, Northhampton, MA, USA).