Photocycle of Cyanobacteriochrome TePixJ

Due to the recent advances in X-ray free electron laser techniques, bilin-containing cyanobacteriochrome photoreceptors have become prime targets for the ever-expanding field of time-resolved structural biology. However, to facilitate these challenging studies, it is essential that the time scales of any structural changes during the photocycles of cyanobacteriochromes be established. Here, we have used visible and infrared transient absorption spectroscopy to probe the photocycle of a model cyanobacteriochrome system, TePixJ. The kinetics span multiple orders of magnitude from picoseconds to seconds. Localized changes in the bilin binding pocket occur in picoseconds to nanoseconds, followed by more large-scale changes in protein structure, including formation and breakage of a second thioether linkage, in microseconds to milliseconds. The characterization of the entire photocycle will provide a vital frame of reference for future time-resolved structural studies of this model photoreceptor.


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output, which has a wavelength of 800 nm, a 1 kHz repetition rate and ~100 fs pulse duration is split, and part of the output is used by an optical parametric amplifier (TOPAS Prime with NIRUVis unit) to generate the excitation beams of 0.6 µJ at 420 nm (for the Pb to Pg reaction) and 0.3 -0.6 µJ at 530 nm (for the Pg to Pb reaction),. The probe beam consisted of a white light continuum generated in a CaF2 crystal by a fraction of the amplifier output. Samples were flowed through a 2 mm pathlength quartz cuvette, where the sample reservoir was illuminated by the LED. The pre-excitation data were subtracted before the analysis and spectral chirp corrected for.
Measurements on µs to s timescales were carried out using a laser system consisting of an optical parametric oscillator (OPO) pumped by a Q-switched Nd: YAG laser (Brilliant B, Quantel).
Excitation wavelengths of 420 and 532 nm were generated using the OPO pumped by the third harmonic of the laser. A quartz cuvette with a 2 mm path length for pump-excitation and 10 mm path length for probing of the was used. TA spectra were recorded using an LKS-60 Flash-Photolysis instrument (Applied Photophysics Ltd.). Kinetics were recorded in 5 nm increments across the visible spectral region.

INFRA-RED TRANSIENT ABSORPTION
Time-resolve infra-red spectroscopy was performed using the TRMPS set-up of the ULTRA-LIFEtime system at the Central Laser Facility, STFC, Rutherford Appleton Laboratory, UK. This uses a 100 kHz ultrafast laser based on a custom dual Yb:KGW system (Pharos, Light Conversion). Samples in D2O buffer were contained between two CaF2 windows, separated by a teflon spacer to give a pathlength of approximately 50 μm. Samples were flowed through the cell, and the sample holder was rastered to avoid sample damage. Excitation beams, at a 1kHz repetition rate were used at wavelengths of 425 nm, with 0.8 µJ pump energy, and 530 nm with 0.6 µJ energy. Data were collected for approximately 40 minutes per dataset. The polarization of the excitation beam was set at the magic angle with respect to the IR probe beam. Difference spectra were generated relative to the ground state at time delays ranging between 1 ps and 390 S4 μs. Pixel to wavenumber calibration was performed using a polystyrene standard. Infra-red pumpprobe transient absorption data from the TRMPS set-up were averaged 0.1 µs on either side of each 10 µs "TRMP" step after the first 10 µs of data. Thus, 58 time points were spread over the first 10 µs timerange, and thereafter timepoints were every 10 µs up to 390 µs (39 in total)

GLOBAL ANALYSIS
Global Analysis was performed using the open-source software Glotaran to obtain an evolution associated difference spectra (EADS). 1 This procedure reduces the matrix of change in absorbance as a function of time and wavelength, to a model of one or more exponentially decaying time components, each with a corresponding difference spectrum (EADS). All datasets were fitted to a sequential, unbranched model.  S7 reactive" ground-state absorption at 575 nm indicates that illumination at 530 nm (which would be absorbed) has not changed the state of the species, confirming it as being non-reactive. The kinetics of the 575 nm bleach returning to the baseline within a nanosecond demonstrates the lack of any long-lived changes after photoexcitation.     Wavenumber (cm -1 ) 0.04 mOD 1548 cm -1 S14 Figure S9. For the Pb to Pg conversion of TePixJ: data points and fitted curves at selected wavelengths/ wavenumbers (indicated in spectra in Figure S8) from global analysis of visible datasets from 0.47 ps to 3.9 µs (A), 0.6 to 450 µs (B, see Figure S10 for more discussion of analysis), and 0.9 to 994 ms (C), and an infrared dataset from 1 ps to 400 µs (D).

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. EADS7 .  Figure S17. For the Pg to Pb conversion of TePixJ: data points and fitted curves at selected wavelengths/ wavenumbers (indicated in spectra in Figure S16) from global analysis of visible datasets from 0.3 ps to 3.6 µs (A), 1 to 450 µs (B), and 0.1 to 45 ms (C, see Figure S18 for more discussion of analysis), and an infrared dataset from 1 ps to 400 µs (D).