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Protonation Heterogeneity Modulates the Ultrafast Photocycle Initiation Dynamics of Phytochrome Cph1 Julia Kirpich, L. Tyler Mix, Shelley S. Martin, Nathan Clarke Rockwell, J. Clark Lagarias, and Delmar S. Larsen J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b01133 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 6, 2018

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The Journal of Physical Chemistry Letters

Protonation Heterogeneity Modulates the Ultrafast Photocycle Initiation Dynamics of Phytochrome Cph1

Julia S. Kirpich,1 L. Tyler Mix,1 Shelley S. Martin2, Nathan C. Rockwell,2 J. Clark Lagarias, 2 and Delmar S. Larsen1*

1

Department of Chemistry University of California, Davis One Shields Ave, Davis, 95616 2

Department of Molecular and Cell Biology University of California, Davis One Shields Ave, Davis, CA, 95616

Running title: Cph1 pH dependence of Pr dynamics

* Corresponding Author: Delmar S. Larsen, Department of Chemistry, University of California, Davis, Davis CA 95616. Telephone: (530) 754-9075; E-mail: [email protected] 1

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ABSTRACT Phytochromes proteins utilize ultrafast photoisomerization of a linear tetrapyrrole chromophore to detect the ratio of red to far-red light. Femtosecond photodynamics in the PAS-GAF-PHY photosensory core of the Cph1 phytochrome from Synechocystis sp. PCC6803 (Cph1Δ) were resolved with a dualexcitation-wavelength-interleaved pump-probe (DEWI) approach with two excitation wavelengths (600 nm and 660 nm) at three pH values (6.5, 8.0, and 9.0). Observed spectral and kinetic heterogeneity in the excited-state dynamics were described with a self-consistent model comprised of three spectrally distinct populations with different protonation states (Pr-I, Pr-II, and Pr-III), each composed of multiple kinetically distinct sub-populations. Apparent partitioning among these populations is dictated by pH, temperature, and excitation wavelength, Our studies provide insight into photocycle initiation dynamics at physiological temperatures, implicate the low-pH/low temperature Pr-I state as the photoactive state in vitro, and implicate an internal hydrogen-bonding network in regulating photochemical quantum yield.

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Emerging genetically encoded biological materials allow researchers to control biological activity with light at the cellular or sub-cellular level.1-7 A common approach in developing such optogenetic materials involves fusing existing light-sensing domains to non-native output domains (e.g., histidine kinase domains or DNA binding proteins) to make novel hybrid photoreceptors. A detailed understanding of the mechanisms that couple light to biological function in existing photoreceptor proteins aids in the engineering of these optogenetic tools. Of the known photoreceptor families, bilinbinding proteins such as phytochromes have attracted interest for such efforts because of their response to long wavelengths that penetrate deeper into mammalian tissue.8 Phytochromes are widespread photoreceptors using covalently attached linear tetrapyrrole (bilin) chromophores to switch between red-absorbing Pr and far-red absorbing Pfr states (Fig. 1A–B).9-12 The N-terminal PAS-GAF-PHY photosensory core module of full-length Cph1 (amino acids 1–514, hereafter termed Cph1Δ) from the cyanobacterium Synechocystis sp. PCC6803 is one of the most well studied model systems for exploring phytochrome photodynamics due to its robust recombinant expression and known crystal structure (Fig. 1C).13-15 Full-length Cph1 includes a C-terminal histidine kinase domain, but Cph1 and Cph1Δ exhibit nearly identical fluorescence spectra, activation barriers for the Pr photoreaction, and photodynamics.16 Red illumination of the dark-adapted Pr state of Cph1Δ (Fig. 1B; red curve) initiates forward photoconversion of the phycocyanobilin (PCB) chromophore (Fig. 1A) to generate the isomerized Lumi-R primary photoproduct on a picosecond timescale.17-22 Lumi-R evolves on the electronic ground-state surface via several intermediates to generate Pfr (Fig. 1B; dark red curve) on a >100 ms timescale.23, 24 The dark-adapted Pr state is then regenerated from Pfr either by farred light (~700 nm) or via thermal reversion (>24 h).9, 13, 25

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Figure 1. (A) The phycocyanobilin (PCB) chromophore of Cph1 is shown in the 15Z (right) and 15E (left) conformations. (B) Absorption spectra are shown for dark-adapted Pr with a 15Z PCB chromophore (red) and for calculated Pfr with a 15E PCB chromophore (dark red) at pH 8.26 (C) The crystal structure showing PAS-GAF-PHY domain architecture of Cph1.15 Under physiological conditions, most proteins continually flex, rotate and sample different metastable states on a complex multi-dimensional potential energy surface.27,

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For phytochromes, as for

photoreceptors in general, these dynamics result in multiple co-existing populations that often exhibit distinct spectra (“spectral heterogeneity”) and/or distinct kinetics (“temporal heterogeneity”). Both types of heterogeneity reflect structural differences in the chromophore itself, such as in protonation state or facial disposition of the bilin rings,29 and/or in the surrounding protein scaffold, such as sidechain conformation30 or protonation state.31 Through analysis of the temperature dependence and excitation wavelength dependence of Cph1Δ fluorescence, Sineshchekov and coworkers provided evidence for the presence of multiple co-existing Pr populations.16 Using variants of Cph1Δ incorporating specific mutations, they argued that 4


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photochemical quantum yield correlates with differences in the relative activation barriers for bifurcating processes on the excited state surface or for partitioning between different sub-populations with different photoactivities.32, 33 This interpretation assumes that the Pr ground state is spectrally homogenous in solution.34 However, Matysik and co-workers used magic angle NMR measurements of Cph1Δ at 243 K and pH 8 to resolve two co-existing Pr populations (Pr-I and Pr-II) with different hydrogen bonding networks around the embedded PCB chromophore.23 Heyne and co-workers similarly argued for inhomogeneity in Cph1 based on polarized transient infrared signals.35 This work demonstrated the existence of structural heterogeneity in the Pr ground state (i.e., without illumination). Spectral heterogeneity has also been observed in wild-type Cph1 by examining the wavelength and temperature dependence of static and ultrafast transient absorption (TA) signals for Cph1Δ at pH 8, photo

revealing two co-existing Pr populations denoted

Pr and

fluor

Pr.36 Excitation of each of these

populations yielded multiple spectrally identical excited-state Pr* sub-populations that decayed on different timescales (temporal heterogeneity), with Pr* sub-populations arising from being spectrally distinct from those arising from

fluor

of Cph1Δ corresponds well with the extracted

photo

Pr excitation

Pr excitation. The fluorescence excitation spectrum fluor

Pr static spectrum, so the room-temperature

fluorescence spectrum of Cph1Δ was assigned to this species.36 This interpretation led to the conclusion that only one

photo

Pr sub-population was almost exclusively responsible for generation of the Lumi-R

primary photoproduct. Remarkably, this ground state heterogeneity is dependent on a temperaturedependent dynamic equilibrium: the longer-living

fluor

Pr population is favored at elevated temperatures,

resulting in inverted Årrhenius behavior for photochemistry.36 Such behavior is difficult to reconcile with a single homogeneous Pr ground state. Further support for a heterogeneous Pr ground state was obtained in a recent study of the pH dependence of the static absorption and resonance Raman spectra of Cph1∆,37 which identified three populations 5



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with differing pH-dependent amplitudes that were ascribed to different intra-protein hydrogen-bonding networks: •

Pr-I that dominates at low pH values ( Pr Photoconversion by Magic-Angle Spinning NMR Spectroscopy (vol 132, pg 4431, 2010). J. Am. Chem. Soc. 2010, 132, 9219-9219. Kim, P. W., Rockwell, N. C., Martin, S. S., Lagarias, J. C., Larsen, D. S. Heterogeneous Photodynamics of the Pfr State in the Cyanobacterial Phytochrome Cph1. Biochemistry 2014, 53, 4601-4611.

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Bu, Z., Callaway, D. J. E. Chapter 5 - Proteins MOVE! Protein dynamics and long-range allostery in cell signaling, In Advances in Protein Chemistry and Structural Biology (Donev, R., Ed.), pp 163-221, Academic Press, 2011. Frauenfelder, H., Chen, G., Berendzen, J., Fenimore, P. W., Jansson, H., McMahon, B. H., Stroe, I. R., Swenson, J., Young, R. D. A unified model of protein dynamics. Proc. Natl. Acad. Sci. USA 2009, 106, 5129-5134. Rockwell, N. C., Duanmu, D., Martin, S. S., Bachy, C., Price, D. C., Bhattacharya, D., Worden, A. Z., Lagarias, J. C. Eukaryotic algal phytochromes span the visible spectrum. Proc. Natl. Acad. Sci. USA 2014, 111, 3871-3876. Lim, S., Yu, Q., Gottlieb, S. M., Chang, C.-W., Rockwell, N. C., Martin, S. S., Madsen, D., Lagarias, J. C., Larsen, D. S., Ames, J. B. Correlating structural and photochemical heterogeneity in cyanobacteriochrome NpR6012g4. Proc. Natl. Acad. Sci. USA 2018, 115, 4387-4392. Hirose, Y., Rockwell, N. C., Nishiyama, K., Narikawa, R., Ukaji, Y., Inomata, K., Lagarias, J. C., Ikeuchi, M. Green/red cyanobacteriochromes regulate complementary chromatic acclimation via a protochromic photocycle. Proc. Natl. Acad. Sci. USA 2013, 110, 4974-4979. Mailliet, J., Psakis, G., Feilke, K., Sineshchekov, V., Essen, L.-O., Hughes, J. Spectroscopy and a HighResolution Crystal Structure of Tyr263 Mutants of Cyanobacterial Phytochrome Cph1. J. Mol. Biol. 2011, 413, 115-127. Sineshchekov, V., Mailliet, J., Psakis, G., Feilke, K., Kopycki, J., Zeidler, M., Essen, L.-O., Hughes, J. Tyrosine 263 in Cyanobacterial Phytochrome Cph1 Optimizes Photochemistry at the prelumi-R→lumi-R Step. Photochem. Photobiol. 2014, 90, 786-795. Spillane, K. M., Dasgupta, J., Lagarias, J. C., Mathies, R. A. Homogeneity of Phytochrome Cph1 Vibronic Absorption Revealed by Resonance Raman Intensity Analysis. J. Am. Chem. Soc. 2009, 131, 13946-13948. Yang, Y., Linke, M., von Haimberger, T., Matute, R., González, L., Schmieder, P., Heyne, K. Active and silent chromophore isoforms for phytochrome Pr photoisomerization: An alternative evolutionary strategy to optimize photoreaction quantum yields. Struct. Dynam. 2014, 1, 014701. Kim, P. W., Rockwell, N. C., Martin, S. S., Lagarias, J. C., Larsen, D. S. Dynamic inhomogeneity in the Photodynamics of Cyanobacterial Phytochrome Cph1. Biochemistry 2014, 53, 2818-2826. Velazquez Escobar, F., Lang, C., Takiden, A., Schneider, C., Balke, J., Hughes, J., Alexiev, U., Hildebrandt, P., Mroginski, M. A. Protonation-Dependent Structural Heterogeneity in the Chromophore Binding Site of Cyanobacterial Phytochrome Cph1. J. Phys. Chem. B 2017, 121, 47-57. Kim, P. W., Rockwell, N. C., Freer, L. H., Chang, C.-W., Martin, S. S., Lagarias, J., Clark, Larsen, D. S. Unraveling the Primary Isomerization Dynamics in the Cph1 Cyanobacterial Phytochrome with Multipulse Manipulations. J. Phys. Chem. Lett. 2013, 4, 2605-2609. Bizimana, L. A., Epstein, J., Brazard, J., Turner, D. B. Conformational Homogeneity in the Pr Isomer of Phytochrome Cph1. J. Phys. Chem. B 2017, 121, 2622-2630. Larsen, D. S., Papagiannakis, E., van Stokkum, I. H. M., Vengris, M., Kennis, J. T. M., van Grondelle, R. Excited state dynamics of beta-carotene explored with dispersed multi-pulse transient absorption. Chem. Phys. Lett. 2003, 381, 733-742. Kim, P. W., Freer, L. H., Rockwell, N. C., Martin, S. S., Lagarias, J. C., Larsen, D. S. Femtosecond Photodynamics of the Red/Green Cyanobacteriochrome NpR6012g4 from Nostoc punctiforme. 2. Reverse Dynamics. Biochemistry 2012, 51, 619-630. Gottlieb, S. M., Kim, P. W., Rockwell, N. C., Hirose, Y., Ikeuchi, M., Lagarias, J. C., Larsen, D. S. Primary photodynamics of the green/red-absorbing photoswitching regulator of the chromatic adaptation E domain from Fremyella diplosiphon. Biochemistry 2013, 52, 8198-8208. Hahn, J., Strauss, H. M., Landgraf, F. T., Gimenez, H. F., Lochnit, G., Schmieder, P., Hughes, J. Probing protein-chromophore interactions in Cph1 phytochrome by mutagenesis. FEBS J. 2006, 273, 1415-1429. Thyrhaug, E., Žídek, K., Dostál, J., Bína, D., Zigmantas, D. Exciton Structure and Energy Transfer in the Fenna–Matthews–Olson Complex. J. Phys. Chem. Lett. 2016, 7, 1653-1660. 22

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Tokmakoff, A. Two-Dimensional Line Shapes Derived from Coherent Third-Order Nonlinear Spectroscopy. J. Phys. Chem. A 2000, 104, 4247-4255. Rockwell, N. C., Shang, L., Martin, S. S., Lagarias, J. C. Distinct classes of red/far-red photochemistry within the phytochrome superfamily. Proc. Natl. Acad. Sci. USA 2009, 106, 6123-6127. Song, C., Psakis, G., Lang, C., Mailliet, J., Gartner, W., Hughes, J., Matysik, J. Two ground state isoforms and a chromophore D-ring photoflip triggering extensive intramolecular changes in a canonical phytochrome. Proc. Natl. Acad. Sci. USA 2011, 108, 3842-3847. Mroginski, M. A., von Stetten, D., Escobar, F. V., Strauss, H. M., Kaminski, S., Scheerer, P., Gunther, M., Murgida, D. H., Schmieder, P., Bongards, C., et al. Chromophore Structure of Cyanobacterial Phytochrome Cph1 in the Pr State: Reconciling Structural and Spectroscopic Data by QM/MM Calculations. Biophys. J. 2009, 96, 4153-4163. Groenhof, G., Schäfer, L. V., Boggio-Pasqua, M., Grubmüller, H., Robb, M. A. Arginine52 Controls the Photoisomerization Process in Photoactive Yellow Protein. J. Am. Chem. Soc. 2008, 130, 3250-3251. Fischer, A. J., Lagarias, J. C. Harnessing phytochrome's glowing potential. Proc. Natl. Acad. Sci. USA 2004, 101, 17334-17339. Toh, K. C., Stojkovic, E. A., van Stokkum, I. H. M., Moffat, K., Kennis, J. T. M. Proton-transfer and hydrogen-bond interactions determine fluorescence quantum yield and photochemical efficiency of bacteriophytochrome. Proc. Natl. Acad. Sci. USA 2010, 107, 9170-9175. Mathes, T., Ravensbergen, J., Kloz, M., Gleichmann, T., Gallagher, K. D., Woitowich, N. C., St. Peter, R., Kovaleva, S. E., Stojković, E. A., Kennis, J. T. M. Femto- to Microsecond Photodynamics of an Unusual Bacteriophytochrome. J. Phys. Chem. Lett. 2015, 6, 239-243.

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Figure 1. (A) The phycocyanobilin (PCB) chromophore of Cph1 is shown in the 15Z (right) and 15E (left) conformations. (B) Absorption spectra are shown for dark-adapted Pr with a 15Z PCB chromophore (red) and for calculated Pfr with a 15E PCB chromophore (dark red) at pH 8.26 (C) The crystal structure showing PAS-GAF-PHY domain architecture of Cph1.15 139x78mm (300 x 300 DPI)


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Figure 2. (A) Normalized static absorption spectra are shown for Cph1∆ in the Pr state at pH 6.5 (dark red curve), pH 8.0 (orange curve) and pH 9.0 (blue curve). (B) Detailed view of the peak region. 269x98mm (290 x 290 DPI)



Figure 3. Comparison of the DEWI TA spectra of Cph1∆ 1 ps after excitation at 600 nm (blue curves) and 660 nm (red curves) at pH 6.5 (A), pH 8.0 (B) and pH 9.0 (C). The spectra obtained at 600 nm excitation are scaled to the amplitude of the band at 660-675 nm of the spectra obtained at 660 nm excitation at pH 8.0. DEWI spectra at 0.5 ps and 5 ps are presented in Figs. S1 and S2. 147x267mm (300 x 300 DPI)


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Figure 4. Integrated target model for DEWI TA signals at different pH values (6.5, 8.0, and 9.0). Pr populations Pr-I, Pr-II, and Pr-III are colored teal, green, and purple, respectively. The relative occupancies of initially excited populations under different experimental conditions are shown in Table S1. The pKa values are estimated from Escobar et al.37 380x70mm (205 x 205 DPI)



Figure 5. Comparison of SADS for different excited-state Pr populations: photoactive (Pr-I*, teal curve), fluorescent (Pr-II*, green curve) and deprotonated (Pr-III*, purple curve). The model is summarized in Fig. 4 with parameters in Table S1. 88x56mm (300 x 300 DPI)


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Figure 6. Simulated 2D electronic spectrum at 9 ps calculated using the model proposed in Figs. 4 and 5 with parameters in Table S1. Purple line, the diagonal; filled circles, peak of the bleach/SE band as a function of excitation frequency. The absorption spectra (left curves) and SADS (top curves) are color coded to match the Pr-I and Pr-II populations in the global model of Fig. 4. 241x229mm (300 x 300 DPI)



Figure 7. Chromophore pocket of Cph1 in the proposed Pr-I and Pr–II configurations.15, 47 (A) Model of the proposed hydrogen bonding network (teal dashed lines) formed when His260 is protonated, derived from solid-state NMR and resonance Raman spectroscopy.47 (B) Pr-II crystal structure (2VEA).15 Alternative perspectives are shown in Figs. S8 and S9. 88x43mm (300 x 300 DPI)


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