Molecular Origin of Photoprotection in. Cyanobacteria Probed by Watermarked

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1 Molecular Origin of Photoprotection in Cyanobacteria Probed by Watermarked Femtosecond Stimulated Raman Spectroscopy Yusaku Hontani, 1, Miroslav Kloz, 1,2, Tomáš Polívka, 3 Mahendra K. Shukla 4, Roman Sobotka 4 and John T.M. Kennis* 1 1 Department of Physics and Astronomy, Vrije Universiteit, Amsterdam, The Netherlands 2 ELI-Beamlines, Institute of Physics, Na Slovance 2, Praha 8, Czech Republic 3 Institute of Physics and Biophysics, Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic 4 Centre Algatech, Institute of Microbiology, Academy of Sciences of the Czech Republic, Třeboň, , Czech Republic *corresponding author, j.t.m.kennis@vu.nl Y.H and M.K. contributed equally 1

2 Supporting Information Methods Sample preparation: The HliC protein was purified and prepared as reported previously. 1 The HliC sample at ph 6.5 in 20 mm MES buffer solution including 10 mm CaCl 2, 0.1% β-dm and 25% glycerol was filled in a 2-mm quartz cuvette (100-QS, Hellma Analytics) for time-resolved experiments. The sample absorbance was ~2 and ~0.4 per 2 mm at 480 nm on time-resolved Raman and transient absorption experiments, respectively. A home-built vibrating sample holder was used to avoid irradiation damage during experiments. Baseline-free femtosecond stimulated Raman spectroscopy (FSRS): Femtosecond stimulated Raman experiments were performed with the watermarking baseline-free stimulated Raman setup reported previously. 2-3 Raman pump (800 nm, ~7 µj) and Raman probe (~ nm) were spatiotemporally overlapped at the sample position with the diameter of ~100 µm. Actinic pump (675 nm, 532 or 488 nm, ~280 nj) was focused on the sample to the diameter of ~150 µm with a time delay from -10 ps to 200 ps. The probe light was dispersed by a spectrograph (Acton SpectraPro SP-2500, Princeton Instruments) and detected by a 1024-pixel back-thinned FFT-CCD detector (S , Hamamatsu). A narrow-band ( ~10 nm) interference filter was used for each excitation wavelength. In 675-nm excitation experiments, 17 time points were measured. In 532- and 488-nm excitation experiments, 25 time points were measured. The sample exposure time to the beams was ~2400 s for 675 nm excitation and ~3000 s for 532- and 488- nm excitation in total for each FSRS experiment. The instrument response function had a width of 100 fs. Transient absorption spectroscopy: Femtosecond transient absorption measurements were performed with a pump-probe setup as reported previously 4-5. A sapphire plate was used for supercontinuum white light generation, and selected wavelength regions; nm were detected by the photodiode array. The time delay was varied up to 2 ns at 95 data points with the minimum temporal step of 20 fs. The diameters of 2

3 the pump and the probe beams at the sample position were ~240 µm and ~90 µm, respectively. The wavelength of the pump beam was centered at 675, 532 or 488 nm with a narrow-band ( ~10 nm) interference filter, and the power was attenuated to ~300 nj. The instrumentresponse function was ~70 fs, estimated from global analysis. Global analysis methodology: Global analysis was performed for the transient absorption spectra using the Glotaran program 5-6. With global analysis, all wavelengths were analyzed simultaneously with a set of common time constants 7. A kinetic model was applied consisting of sequentially interconverting, evolutionassociated difference spectra (EADS), i.e in which the arrows indicate successive monoexponential decays of a time constant, which can be regarded as the lifetime of each EADS 7. The first EADS corresponds to the difference spectrum at time zero. The first EADS evolves into the second EADS with time constant τ 1, which in turn evolves into the third EADS with time constant τ 2, etc. The procedure clearly visualizes the evolution of the intermediate states of the protein 8. Decay-associated difference spectra (DADS) indicate the spectral changes with parallel decay channels and independent decay time constants. It is important to note that parallel and sequential analysis are mathematically equivalent and yield identical time constants 9. The standard errors in time constants were less than 5% 6-8. References 1. Shukla, M. K.; Llansola-Portoles, M. J.; Tichy, M.; Pascal, A. A.; Robert, B.; Sobotka, R. Binding of Pigments to the Cyanobacterial High-Light-Inducible Protein HliC. Photosynth. Res Kloz, M.; Weissenborn, J.; Polivka, T.; Frank, H. A.; Kennis, J. T. M. Spectral Watermarking in Femtosecond Stimulated Raman Spectroscopy: Resolving the Nature of the Carotenoid S* State. Phys. Chem. Chem. Phys. 2016, 18 (21), Hontani, Y.; Inoue, K.; Kloz, M.; Kato, Y.; Kandori, H.; Kennis, J. T. M. The Photochemistry of Sodium Ion Pump Rhodopsin Observed by Watermarked Femto- to Submillisecond Stimulated Raman Spectroscopy. Phys. Chem. Chem. Phys. 2016, 18 (35), Hontani, Y.; Shcherbakova, D. M.; Baloban, M.; Zhu, J.; Verkhusha, V. V.; Kennis, J. T. Bright Blue-Shifted Fluorescent Proteins with Cys in the GAF Domain Engineered from Bacterial Phytochromes: fluorescence mechanisms and excited-state dynamics. Sci. Rep. 2016, 6, Ravensbergen, J.; Abdi, F. F.; van Santen, J. H.; Frese, R. N.; Dam, B.; van de Krol, R.; Kennis, J. T. M. Unraveling the Carrier Dynamics of BiVO4: A Femtosecond to Microsecond Transient Absorption Study. J. Phys. Chem. C 2014, 118 (48),

4 6. Snellenburg, J. J.; Laptenok, S. P.; Seger, R.; Mullen, K. M.; van Stokkum, I. H. M. Glotaran: A Java-Based Graphical User Interface for the R Package TIMP. J. Stat. Softw. 2012, 49 (3), van Stokkum, I. H.; Larsen, D. S.; van Grondelle, R. Global and Target Analysis of Time- Resolved Spectra. Biochim. Biophys. Acta 2004, 1657 (2-3), Kennis, J. T. M.; Groot, M. L. Ultrafast Spectroscopy of Biological Photoreceptors. Curr. Opin. Struc. Biol. 2007, 17 (5), Toh, K. C.; Stojkovic, E. A.; van Stokkum, I. H.; Moffat, K.; Kennis, J. T. Fluorescence Quantum Yield and Photochemistry of Bacteriophytochrome Constructs. Phys. Chem. Chem. Phys. : Phys. Chem. Chem. Phys. 2011, 13 (25),

5 0.5 -GS Raman (a.u.) FSRS, 2.5 ps 0.0 mod Wavenumber (cm -1 ) Figure S1. FSRS of a Chl a β-carotene mixture in tetrahydrofuran (THF). Red line: FSRS spectrum at 2.5 ps upon excitation at 675 nm. Black line: ground state stimulated Raman spectrum, inverted and scaled to the red line on the 1520 cm -1 β- carotene C=C stretch. The bands at 1580 and 1690 cm -1 arise from Chl a, whereas the bands at 1430 and 1480 cm -1 arise from the THF solvent. 5

6 Figure S2. Transient absorption spectrum of HliC upon 675-nm excitation. EADS and DADS of (A) nm and (B) nm spectral regions. 6

7 Figure S2. Transient absorption kinetic trace at 580 nm (magenta open dots) with a fitting curve (magenta line) overlapped with FSRS data at 1774 cm -1 (black closed squares) on a 200 ps timebase. The excitation wavelength was 675 nm. 7

8 Figure S4. FSRS of HliC with β-car excitation. Upon excitation at (A) 488 nm and (B) 532 nm. 8

9 Figure S5. Transient absorption spectra of HliC with β-car excitation. Upon excitation at (A) 488 nm and (B) 532 nm. Spectral region of nm was omitted in (B) because of the strong pump light scattering. 9

10 Figure S6. Comparison of FSRS peaks of HliC on the S1 state of β-car upon excitation at different wavelengths. Signals upon excitation at 488, 532 and 675 nm are shown in cyan, green and red, respectively. 10