PigmentâProtein Interactions in Phytochromes Probed by

Article pubs.acs.org/JPCB

Pigment−Protein Interactions in Phytochromes Probed by Fluorescence Line Narrowing Spectroscopy Jana B. Nieder,†,# Emina A. Stojković,‡,§ Keith Moffat,‡ Katrina T. Forest,⊥ Tilman Lamparter,∥ Robert Bittl,† and John T. M. Kennis*,¶ †

Fachbereich Physik, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany Department of Biochemistry and Molecular Biology, Center for Advanced Radiation Sources, and Institute for Biophysical Dynamics, University of Chicago, 920 East 58th Street, Chicago, Illinois 60637, United States ⊥ Department of Bacteriology, University of WisconsinMadison, 1550 Linden Drive, Madison, Wisconsin 53706, United States ∥ Botany 1, KIT - Karlsruhe Institute of Technology, Kaiserstrasse 2, D 76131 Karlsruhe, Germany ¶ Department of Physics and Astronomy, Biophysics Section, VU University Amsterdam, De Boelelaan 1081, NL-1081 HV Amsterdam, The Netherlands ‡

ABSTRACT: Fluorescence line narrowing (FLN) spectroscopy was used to study bacteriophytochromes and variants from various species in their red-absorbing Pr ground state, including phytochromes Agp1 from Agrobacterium tumefaciens, DrBphP from Deinococcus radiodurans, and RpBphP2 and RpBphP3 from Rhodopseudomonas palustris. A species-dependent narrowing of the fluorescence emission bands is observed. The results suggest varied pigment−protein interactions, possibly connected to chromophore mobility or extended water pyrrole networks inside of the differing binding pockets. Solvent water isotope exchange from H2O-based buffer to D2O-based buffer solutions was used to identify specific vibrational modes of the chromophore. In addition to the expected frequency shifts upon isotope exchange, the line narrowing efficiency is increased in deuterated compared to protonated surroundings. We conclude that proton dynamics inside of the protein binding pocket are a dominant source of spectral diffusion at low temperatures, which possibly relates to the previous observation that the electronic transition is directly coupled to proton transfer. The FLN spectra of Agp1 reconstituted with a synthesized pigment shows strong line narrowing efficiency even in protonated buffer solution. The FLN spectra of a point mutant of RpBphP3 highlight the involvement of aspartate 216 in a hydrogen bond network around the chromophore. On the basis of similar FLN characteristics in RpBphP2 and RpBphP3, we propose a similarly extended hydrogen bond network around their chromophores despite the different photoreactions leading to red- or blue-shifted absorption relative to the respective photoreceptors’ ground-state absorption.

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INTRODUCTION The red and far-red light-sensitive phytochromes are used by many organisms such as plants, bacteria, and fungi to grow and develop in a light-dependent manner.1−3 In plants, processes such as shade avoidance, seedling development, germination, and flowering are controlled by phytochrome signaling. Phytochromes covalently bind an open-chain tetrapyrrole from the bilin family. Buried inside of the phytochrome binding pocket, the tetrapyrrole is stabilized in two isomeric forms, the Pr form absorbing in the red and the Pfr form absorbing in the far-red spectral range (Figure 1a). These conformers are reversibly interconvertible by illumination with red and far-red light, respectively. The primary, photoinduced conformational change of the pigment occurs within picoseconds and is followed by a signal cascade comprising conformational changes of the pigment binding pocket and those of more distant regions of the protein,4 in which the tertiary and quaternary structure of the protein changes with time constants of micro- to milliseconds.5,6 Figure 1b shows the © 2013 American Chemical Society

absorption spectra of the bacteriophytochrome Agp1 before and after irradiation with red light. The main absorption peak before illumination with red light is found at 700 nm, but after illumination, a new spectral contribution rises at 750 nm. A number of different intermediates in the course of the forward and backward reactions between Pr and Pfr were trapped and spectrally characterized at low temperature.7−17 The tetrapyrrole chromophore in the Pr and Pfr states was found to be fully protonated11,18,19 but underwent de- and reprotonation steps during the photoreaction.18 Structural models show that phytochromes enclose bulk water around their open-chain tetrapyrrole chromophore, forming a so-called pyrrole−water network via hydrogen bonds (see Figure 1c).20 In comparison with the pigment conformation found for phytochromes in the Pr state,20−22 the crystal structure of the bacteriophytochrome Received: September 11, 2013 Revised: November 8, 2013 Published: November 8, 2013 14940

dx.doi.org/10.1021/jp409110q | J. Phys. Chem. B 2013, 117, 14940−14950

The Journal of Physical Chemistry B

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gene photoswitches, fluorescent reporters, and functional materials in agricultural biotechnology.36−43 The near-infrared-absorbing phytochromes are excellent candidates for increasing the range of imaging applications particularly in deeper-lying mammalian tissues. However, for this purpose, the fluorescence quantum yield needs to be artificially increased above that of native phytochromes. Different strategies tested on various bacteriophytochromes include insertion of fluorescent dyes44,45 and modifications of the chromophore binding pocket.29,33,37,46 These show high potential based on recent advances in the understanding of competing deactivation processes.29 Bacteriophytochromes are characterized by structural and spectroscopic variability.47,48 Structural heterogeneity of the tetrapyrrole chromophore may be an intrinsic property of phytochromes,4,29,48−50 in either their excited state or ground state.14,29,49,51−54 Ensemble spectroscopic methods imply signal averaging over all subconformers, which renders spectra complex and makes it difficult or even impossible to disentangle the underlying signals. In order to avoid these problems of ensemble spectroscopic methods, selective spectroscopies such as fluorescence single-molecule spectroscopy55 and fluorescence line narrowing (FLN)56 spectrocopy can be applied. To observe line-narrowed fluorescence emission spectra, a narrow band excitation laser is tuned to the red edge of the inhomogeneously broadened absorption profile of the sample,56−60 such that efficient electronic excitation takes place only for a subset of molecules and avoids overlapping fluorescence emission arising from vibrationally excited molecules. Fluorescence single-molecule spectroscopy on phytochrome revealed an extensive dynamic intercomplex heterogeneity of the phytochrome.48 Here, we apply the FLN technique to these bilin-based photoreceptors. We examined in total nine different phytochrome samples. First, three variants of Agp1: (i) Agp1-PCD: this wild-type photosensory core domain (PCD) fragment consists of the PAS-GAF-PHY domains and shows full phytochrome photochromicity;61 (ii) Agp1-PCD-Y166H: a mutant with a tyrosineto-histidine mutation at position 166, located near the chromophore binding site (Figure 1); in the cyanobacterial phytochrome Cph1, the analogous tyrosine-to-histidine mutation generates a species that is strongly fluorescent under ambient conditions;33 (iii) Agp1-PCD-BV15Za: a variant where the native biliverdin (BV) chromophore is replaced by the synthetic chromophore BV15Za, in which an additional covalent bond between ring C and ring D locks the chromophore in a Pr-like conformation.62 We also examined two samples from the bacterium D. radiodurans.: (iv) DrBphP: the wild-type full-length protein and (v) DrBphP-CBD-Y307S: the chromophore binding domain (CBD) containing only the PAS-GAF domains and a serine-to-tyrosine mutation in the protein dimerization interface, far from the chromophore site. This variant shows improved crystallization characteristics compared to wild-type.20 Finally, we examined two different phytochromes and variants from R. palustris: (vi) RpBphP2 fulllength protein and (vii) the unusual and also full-length RpBphP3, which has a regular UV/vis absorption spectrum in the Pr state but produces a blue-shifted Pnr (“near-redabsorbing”) state after photoactivation with red light (at 650 nm).63 A notable difference in the immediate surroundings of the pigment in RpBphP3 compared to that in RpBphP2 is the number of hydrogen bonds between the pigment and its

Figure 1. The photochromic pigment biliverdin (BV) as a cofactor of bacteriophytochromes. (a) Structure of the phytochrome-bound BV in the stable Pr and metastable Pfr form. The putative isomerization along the C15C16 double bond that causes a rotation of pyrrole ring D is visualized. The numbering of the C atoms27 is shown in green. The double bond between C3 and C31 as in ref 20 possibly leads to an extended π-conjugation system in the direction of the covalent cysteine binding site, as indicated by the gray ellipse. (b) Absorption spectra of Agp1 phytochrome in its Pr ground state and after illumination with red light of λexc = 705 ± 5 nm. (c) Locations of aspartate and tyrosine inside of the chromophore binding pocket of DrBphP. A zoom into the binding pocket is given, showing BV in the Pr form (PDB entry: 2O9C).20 The locations of the mutated side chains in RpBphP3-PCD-D216A and Agp1-PCD-Y166H are indicated.

from Pseudomonas aeruginosa in the Pfr state23 corroborates the results from vibrational spectroscopic analyses. These indicate that Z/E isomerization at the C15C16 double bond is the primary photochemical event during the Pr to Pfr photoreaction.12,24−26 While crystallization and structure determination of phytochrome has so far only been successful with phytochrome domains such as the photosensory core module,20−22,28 spectroscopic approaches also allow investigation of the full-length protein including its C-terminal histidine kinase domain. By comparison of spectroscopic data on the native phytochrome system and, specifically, modified phytochromes, the functional influence of specific regions on the photophysical and photochemical processes can be analyzed.29−35 Phytochromes attract an increasing amount of attention as targets for protein engineering due to their potential use as 14941



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surrounding.4,64 In RpBphP3, three hydrogen bonds are formed with neighboring His, Lys, and Ser residues to the ring D carbonyl, while in the canonical bacteriophytochromes such as RpBphP2, only one hydrogen bond is formed with a His residue: (viii) RpBphP3-PCD: displaying photoconversion efficiency comparable to that of the full-length protein22 and (ix) RpBphP3-PCD-D216A: a variant with an aspartate-toalanine mutation near the chromophore binding site at position 216 (see Figure 1d). This mutation influences the photochemistry of phytochromes by changing the hydrogen-bonding and proton-transfer characteristics.29 Samples (vi−ix) from the phytochromes from R. palustris were also analyzed in deuterated buffer, where they are denoted as samples (x−xiv).

of the chromophore BV15Za with Agp1 is described in ref 62, purification of the phytochromes from R. palustris is described in refs 22 and 66, and details for DrBphP from Deinococcus radiodurans (D. radiodurans) are in ref 21. Dilution was accomplished under safe green light conditions for all phytochromes to prevent red-light-inducible photoconversion and preserve their Pr ground state. For the lowtemperature FLN measurements, A. tumefaciens phytochrome and variants were prepared in 50 mM Tris/Cl, 5 mM EDTA, 150 mM NaCl, pH 7.8. R. palustris and D. radiodurans phytochromes and variants, were prepared in 30 mM Tris/Cl, pH 8.0. The 60% glycerol (w/w) allowed transparent glass formation under low-temperature conditions. For deuterated samples, the buffer was exchanged to D2O-based buffer solutions, which were adjusted by DCl and using the relation pD = pH + 0.4. Exchange was done by centrifuging the sample in an a > 30 kDa microfilter at a speed of 9000 rpm for 15 min. The concentrated protein was then diluted in the D2O-based buffer, and the procedure repeated three times, followed by addition of deuterated glycerol to a concentration of 60% (w/ w). UV/vis spectra were measured for all samples. The optical density and extinction coefficients at 280 nm yield the sample concentration. Extinction coefficients, absorption maxima, and the experimental parameters such as excitation wavelength and acquisition times used for FLN measurements are given in Table 1. An exchange chamber allowed sample exchange at low temperature. For each FLN spectrum, the laser excitation wavelength position was used as the origin of the relative wavenumber scale. Peak positions are determined for the various phytochrome variants by applying the “max” function in MATLAB67 in selected wavenumber ranges around the peaks. The values are given in wavenumbers with an estimated error of ±6 cm−1, mainly due to the spectral resolution of 0.2 nm. FLN spectra are all given on a relative wavenumber scale with respect to the excitation wavelength. For all FLN measurements, the excitation power was measured in front of the cryostat.

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MATERIALS AND METHODS The FLN setup was as described earlier.60 In short, a tunable continuous-wave titanium:sapphire laser pumped by an argon ion laser (Coherent Inc., Santa Clara, U.S.A.) was used as the source, providing variable excitation wavelengths between 700 and 1030 nm with a narrow bandwidth of ∼0.1 cm−1. A standard fluorescence detection setup working in 90° geometry was used. The sample was placed inside of the helium flow cryostat that provided temperatures between 4.2 and 300 K (UTRECS; Cryogenic Technologies Laboratory, Kiev, Ukraine). For spectral analysis, a spectrograph (500is; Chromex, Albuquerque, U.S.A.) with a 600 lines per mm grating and a CCD camera (ST-6, Santa Barbara Instrument Group, Santa Barbara, U.S.A.) were used. CCD chips can be affected by cosmic rays hitting the camera chip. These lead to sharp highintensity bursts. Negative spikes are are due to damaged pixels. Both artifacts affect one or a few pixels only and therefore can easily be identified in the FLN spectra, where the vibrational zero phonon lines (vZPLs) are homogenously and inhomogenously broadened. Hence, steep 1 pixel contributions were not taken into account for the analysis. The resolution of the setup was 0.2 nm. All phytochrome samples analyzed are listed in Table 1. Details of protein expression, extraction, and purification of the phytochrome Agp1 from Agrobacterium tumefaciens (A. tumefaciens) are described in ref 65; the synthesis and assembly

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RESULTS Figure 2 shows the temperature-dependent fluorescence emission spectra of Agp1-PCD-BV15Za at temperatures

Table 1. Absorption Maxima λabs of the Phytochrome Variants Measured by UV/Vis Spectroscopy and Measurement Parameters Used for FLN Spectroscopy, Including the Excitation Wavelength λexc, the Laser Power PL as Measured in Front of the Cryostat, and the Acquisition Time tacq Used to Collect the Spectrum phytochrome sample

solvent

λabs nm

λexc nm

PL μW

tacq s

Agpl-PCD-BV Agpl-PCD-Y166H-BV Agpl-PCD-BV15Za RpBphP2-BV RpBphP3-BV RpBphP3-CBD-BV RpBphP3-D216A-BV RpBphP2-BV RpBphP3-BV RpBphP3-CBD-BV RpBphP3-D216A-BV DrBphP-BV DrBphP-CBD-Y307S-BV

H2O H2O H2O H2O H2O H2O H2O D2 O D2 O D2 O D2 O H2O H2O

702 703 714 710 704 703 704 709 704 703 703 703 702

714 718 726 720 708 711 710 720 712 710 712 715 718

200 100 100 100 100 100 100 100 100 100 100 100 100

100 379 32 240 30 30 180 100 85 96 28 120 46

Figure 2. Temperature-dependent fluorescence emission spectra of Agp1-PCD-BV15Za taken under FLN excitation with λexc = 726 nm and PL = 100 μW; acquisition times were 4 s at 6 K and 1 s at other temperatures.

between 59 and 6 K. For Agp1-PCD-BV15Za with its maximum absorption in the red at 714 nm, the excitation wavelength for FLN spectroscopy was adjusted to 726 nm to ensure that only a subensemble of molecules with extreme redshifted electronic energies gets excited via their 0−0 transition. For the spectrum taken at 59 K, a maximum is found at 144 14942



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Table 2. Peak Positions in the Extended Fingerprint Region from 700 to 1800 cm−1 Determined from the 6 K FLN Spectra Taken on the Phytochromes Agp1-PCD, Agp1-PCD-Y166H, Agp1-PCD-BV15Za, DrBphP, DrBphP-CBD-Y307S, RpBphP2, RpBphP3, RpBphP3-PCD, and RpBphP3-PCD-D216A along with Results from FTIR Spectroscopy (from ref 32) and RR Spectroscopy (from ref 35) for the Phytochromes from R. palustris (left) for Protonated Samples and (right) for Deuterated Samplesa

a Peak positions are given with an error of ±6 cm−1. The peak positions with a clear kinetic isotope effect are highlighted by gray boxes. Strong peak intensities are indicated by a superscript “S”.

cm−1. This maximum shifts to lower frequencies upon decreasing temperature, to 120 cm−1 at 6 K. This temperature-dependent spectral contribution can be identified as the phonon wing (PW) corresponding to the 0−0 transition, which was not included in the detection range. At 59 K, the fluorescence emission spectrum is broad and relatively unstructured. At higher wavenumbers, a shoulder is found at ∼300 cm−1 and two further peaks at 1300 and 1600 cm−1. The broad fluorescence emission extends to about 1820 cm−1, above which the noise level is reached. At lower temperatures, the line width decreases, and vibrational fine structure with a line width of ∼10 cm−1 is superimposed on the broader fluorescence bands. About 40 of these narrow peaks are resolved at 6 K. The vibrational fine structure does not show a temperaturedependent wavenumber shift.

The narrow lines are attributed to the ground-state vibrational modes of the chromophore coupling to the S1,ν=0 to S0,νi electronic transition and are commonly referred to as vibrational zero phonon lines (vZPLs). These vibrational transitions can couple to delocalized phonon modes, leading to red-shifted PWs associated with the vZPLs. In the 6 K spectrum, PWs are observed red-shifted to the narrow vZPLs located at ∼300, 820, 1300, and 1610 cm−1. For other vZPLs, the associated PWs are not clearly observed, perhaps due to the high density of narrow peaks in several ranges of the spectrum or to lower coupling strength of the associated vibrational transitions to phonon modes. FLN spectra were taken for all samples at 6 K and are shown in the Figures 3−5. The excitation wavelength was selected for each sample individually, balancing between the signal intensity 14943



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fluorescence emission signals on the single-molecule level on Agp1.48 Only nine narrow substructures with low relative intensity are found on the high-energy side of the broadened bands with widths of ∼100 cm−1. Broad fluorescence emission bands are observed at ∼830, 1400, and 1670 cm−1 for Agp1PCD, while the middle band is shifted to 1350 cm−1 for Agp1PCD-Y166H. In marked contrast to these results, the variant Agp1-PCDBV15Za reconstituted with the synthetic chromophore shows a highly structured FLN spectrum (as already presented in the temperature-dependent series). In the extended fingerprint region, 18 vZPLs and fine structures were determined, with the most prominent peaks at 744, 824, 903, 996, 1040, 1299, 1315, 1572, and 1608 cm−1. Figure 3 (bottom) shows the fingerprint region of the FLN spectra taken for the full-length protein DrBphP from D. radiodurans and its variant DrBphP-CBD-Y307S. Shifting the laser excitation wavelength to the red edge of the absorption spectrum again did not lead to the observation of vibrationalresolved vZPLs. The degree of spectral diffusion is similar to that in Agp1-PCD and Agp1-PCD-Y166H. Broad fluorescence bands with widths of ∼100 cm−1 are found for both phytochrome samples at ∼820, ∼1400, and ∼1670 cm−1. For DrBphP 10 and for DrBphP-Y307S 7, peak positions were determined. As for earlier samples, the subtle fine structure is mostly observed on the high-energy side of the broadened bands. Figure 4 shows the fingerprint region of the FLN spectra taken from the R. palustris phytochromes and phytochrome variants, scaled to similar intensity in the broad band at around 1650 cm−1. The spectra show high vZPL occurrence compared with the spectra from the wild-type fragment and point mutants of Agp1 and DrBphP samples. For all variants from R. palustris, broad fluorescence emission bands similar to those observed for Agp1 and DrBphP phytochromes are located at around 830, 1360, and 1650 cm−1. These broad bands indicate spectral diffusion effects. The combination of narrow together with broad bands in the FLN spectra from the R. palustris variants indicates heterogeneous spectral diffusion of the molecules. For RpBphP2, RpBphP3, and RpBphP3-PCD, 11, 7, and 9 vZPLs and vibrational substructures were identified in their FLN spectra, respectively. The vZPL with the highest relative intensity is found at 805 cm−1 for RpBphP2. This mode is shifted slightly in RpBphP3 and its variants; a similarly intense peak is present at 816 cm−1 for RpBphP3, 820 cm−1 for RpBphP3-PCD, and 823 cm−1 for RpBphP3-PCD-D216A. The range at around 800 cm−1 is indicative for C−H out-of-plane (HOOP) modes of the tetrapyrrole chromophore.69 The highest occurrence of well-resolved vZPLs from this series on phytochromes and variants from R. palustris is observed for RpBphP3-PCD-D216A, with 20 vZPLs and vibrational substructures. The major spectral difference introduced by the point mutation D216A inside of the chromophore binding pocket is observed on the high wavenumber side of the peak located at 820 cm−1. A broadened band is observed for the wild-type and fragment of RpBphP3, and for the D216A point mutant, a substructure with two peaks located at 840 and 852 cm−1 is resolved. Additionally, two resolved peaks are observed on the high wavenumber side of the peaks located at 750 and 962 cm−1, with maxima at 763 and 780 cm−1 and at 974 and 993 cm−1, respectively.

and evolution of narrow lines, and is listed in Table 1. The covered wavenumber range from 700 to 1800 cm−1 contains the vibrational fingerprint region of tetrapyrroles, which are indicative for the chromophore conformation (1500−1700 cm−1).31,68 Peak positions are given in Table 2. As here, FLN spectroscopy is used for the first time to investigate phytochromes, and the peak positions that we assign to vZPLs are compared to vibrational data obtained with more established techniques, namely, Fourier transform infrared (FTIR) spectroscopy32 and resonance Raman (RR) spectroscopy.35 Figure 3 (top) shows the FLN spectra of the phytochrome variants from A. tumefaciens. The FLN spectra taken from

Figure 3. FLN spectra in the extended fingerprint region taken from Agp1-PCD, Agp1-PCD-Y166H, Agp1-PCD-BV15Za, DrBphP, and DrBphP-CBD-Y307S at 6 K. Further experimental parameters are given in Table 1. The peak positions are given in wavenumbers with an estimated error of ±6 cm−1.

Agp1-PCD and Agp1-PCD-Y166H do not show substantial narrow peaks in the spectral range of 700−1800 cm−1. Spectral diffusion may prevent the evolution of narrow line structures under FLN excitation. For Agp1-PCD (which retains the full photochemical reactivity of the full-length protein), this agrees with the observation of strongly spectral diffusion-affected 14944



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Figure 5. FLN spectra in the extended fingerprint region for RpBphP2 and RpBphP3 as well as from the variants RpBphP3-PCD and RpBphP3-PCD-D216A in deuterated buffer solutions at 6 K. Further experimental parameters are given in Table 1. The peak positions are given in wavenumbers with an estimated error of ±6 cm−1.

Figure 4. FLN spectra in the extended fingerprint region for RpBphP2, RpBphP3, and the variants RpBphP3-PCD and RpBphP3-PCD-D216A, in protonated buffer solutions at 6 K. Further experimental parameters are given in Table 1. The peak positions are given in wavenumbers with an estimated error of ±6 cm−1.

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FLN spectra were also obtained in deuterated buffer solutions for the phytochromes from R. palustris, as shown in Figure 5. The overall spectral shape differs from those in protonated buffer solutions in the ranges of 1040−1250 and 1400−1570 cm−1, where an increase in both broad fluorescence intensity and fine structure are present. Upon deuteration, specific spectral lines arise, and other lines vanish from the spectra. For example, the bands characteristic for protonated samples at around 1320 and 1570 cm−1 are replaced in deuterated samples by bands at ∼1080, 1420, and 1490 cm−1. This isotope effect indicates that deuteration at exchangeable proton sites occurred. In tetrapyrroles, these sites are the pyrrole nitrogens; the carbon sites are less acidic and show low probability for H/D exchange. The H/D exchange at exchangeable proton sites is associated with a lowering of the frequency of the vibrational modes. A peak (which is assumed to stem from the same nitrogen site)18 is located at 1570 cm−1 in H2O and shifts to 1080 cm−1 in deuterated buffer. This deuteration effect is clearly observed in all FLN spectra taken on all phytochrome and phytochrome variants from R. palustris. Upon deuteration, the number of vZPLs on top of the broad fluorescence emission increases significantly. Prominent in the spectra measured in D2O buffer are features occurring in line multiplets comprising four vZPLs. The positions of these multiplets (taken from the spectrum where these are best resolved) are 954/971/987/998 cm−1 for RpBphP3-PCDD216A, 1047/1075/1099/1129 cm−1 for RpBphP3-PCDD216A, 1279/1299/1313/1326 cm−1 for RpBphP3-PCD, and 1431/1446/1468/1493 cm−1 for RpBphP3-PCD.

DISCUSSION We first discuss the species-dependent characteristics of the FLN spectra and then analyze changes in those spectra associated with specific modifications in the binding pockets of the phytochrome chromophore. Phytochrome Species Dependency. The wild-type phytochromes are each well-suited to FLN analysis. Even though all variants were analyzed under closely similar experimental conditions, line narrowing occurred to a different extent in different species. For each sample, the excitation laser wavelength was tuned over the red edge of the absorption band to obtain line narrowing conditions but for Agp1 and DrBphP, but no line narrowing in the form of vZPLs in the fluorescence emission spectra was achieved. Only subtle fine structure is present in the strongly broadened fluorescence emission spectra of these phytochromes. In contrast, for both RpBphP2 and RpBphP3 from R. palustris, FLN was clearly observed. These two phytochromes show fine structure to a similar extent. Even though these phytochromes have substantially different photoreactions at room temperature, their FLN spectra show high similarity. The appearance of substantial fine structure in FLN spectra reveals a rather low degree of spectral diffusion associated with the chromophore. From single-molecule spectroscopy on various pigment−protein complexes including PSI70,71 and LH2 complexes,72 it was deduced that the degree of spectral diffusion, which is dynamic wavelength changes, of a chromophore is specific to its binding conditions. A process relevant to line broadening at low temperatures is proton 14945



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Figure 6. Model of proton dynamics related sources for line broadening, which is observed in fluorescence emission spectra from chromophores at low temperatures. (A) Remaining proton mobility leads to interactions of the chromophore with two-level systems (TLSs) and multi-TLS, which are due to proton dynamics, for example, in hydrogen bridges connecting with the chromophore or nearby water molecules (left panel, taken with modifications from ref 73). (B) In phytochromes, an additional channel, associated with the ESPT step occurring during the photocycle of the photoreceptor, has to be considered when listing sources for spectral diffusion effects (right panel). This excited-state reaction renders phytochromes especially prone to spectral diffusion.

mobility inside of the protein.73 This process is discussed below specifically for the bacteriophytochromes. The FLN spectra indicate that proton mobility in the first coordination sphere of the pigments from Agp1 and DrBphP is higher than that in RpBphP2 and RpBphP3, where vZPLs are well-resolved. The broad spectral bands observed for Agp1 with FLN spectroscopy are in line with observations made on the singlemolecule level.48 Dynamic wavelength changes were observed for individual molecules even at temperatures of 1.4 K. These time-dependent spectral changes can qualitatively be understood in the energy landscape model of proteins.74 For phytochromes, inter- as well as intracomplex heterogeneity was observed with respect to the degree of spectral diffusion of individual phytochrome chromophores.48 This heterogeneity distinguishes the phytochrome tetrapyrrole chromophore from the chromophores found in other pigment−protein complexes, such as PSI71,75 and LH2 complexes.72 Although minor spectral diffusion in a subensemble of phytochrome molecules leads to narrow fluorescence emission peaks containing vibrational information, the majority of Agp1 molecules show a comparatively high degree of spectral diffusion and strongly broadened fluorescence emission bands whose line widths are close to those found for an Agp1 bulk sample.48 For certain individual phytochrome molecules, the vibrational fine structure is substantially better resolved than that achieved here by FLN spectroscopy on Agp1. This can be understood because in FLN spectroscopy, the detected signal is the sum of the emission characteristics belonging to a subensemble of phytochrome molecules. Depending on the intrinsic heterogeneity of the studied system together with its affinity to strong spectral diffusion, the stronger spectral fine structure is averaged out while analyzing more than one molecule at the time. In the crystal structure of DrBphP at 2.521 and 1.45 Å resolution,20 a highly extended hydrogen bond network was identified. In DrBphP, one internal water referred to as the pyrrole water is found close to the BV pigment, within bonding distance of the pyrrole nitrogens of rings, A, B, and C, and close

to a highly conserved aspartate (Asp207 in DrBphP) and histidine (His-260 in DrBphP). For Agp1, the resolution of 3.2 Å is not high enough to locate internal water molecules.76 RpBphP3 crystallized as a dimer with subtle differences between the structures of the monomers. For example, the pyrrole water molecule is only observed in one monomer. If the pyrrole water is absent, hydrogen bonding around BV is largely restricted to interactions with the conserved aspartate (Asp-216 in RpBphP3).29 We propose that this structural difference in the degree of hydrogen bonding is the main cause of the difference in line narrowing. For RpBphP2, a pyrrole water is found, which seems to form slightly different hydrogen bond contacts with the BV pigment compared to the arrangement in RpBphP3.77 Due to the high similarity in line narrowing in RpBphP2 and RpBphP3, we propose that the hydrogen-bonding networks around the pigments in RpBphP2 and RpBphP3 are similar with respect to their ability to facilitate proton dynamics and that their probability of having a pyrrole water near the pigment is small compared to that of DrBphP and Agp1. Phytochrome Mutants. The point mutant DrBphP-CBDY307S (the variant used for improved crystallization characteristics20) did not show differences in FLN compared to wildtype; both showed broadened spectra that lack substantial vibrational information. Because this point mutation is remote from the chromophore site, similar FLN spectra would indeed be expected for these samples. On the other hand, for the point mutant RpBphP3-PCD-D216A in which the mutation is within hydrogen-bonding distance of the pigment, an effect is observed with FLN. The degree of line narrowing is increased in this D216A mutant compared to wild-type. The alteration indicates less proton mobility in the first coordination sphere of the pigment. This conclusion is in line with findings from femtosecond time-resolved absorption spectroscopy, which indicated a less favorable hydrogen-bonding configuration for excited-state proton transfer (ESPT) for this mutant.29 The replacement of aspartate by alanine mutation in the binding pocket leads to a disturbed photoreaction,18 indicating 14946



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the expected broadening behavior at increased temperatures, thereby confirming the origin of the narrow line features as vibrational resolved fluorescence emission lines. The differences in fine structure in the FLN spectra between Agp1-PCD and Agp1-PCD-BV15Za (Figure 3; dark blue and red spectra) indicate a reduced degree of spectral diffusion for the variant with the synthetically locked chromophore. This might arise from a reduced integration into the hydrogen bond network present in the wild-type phytochromes or might be related to the reduced pigment flexibility. The rigid BV15Za is supposed to freeze in a more defined way compared to the open-chain BV chromophore where ring D may assume various (frozen) twist angles along the C15C16 bond. It has been suggested that the twisting of ring D is a prerequisite for the ESPT step.29 In conclusion, the increased probability for ESPT in the more flexible native BV can additionally be a reason for the observed increased spectral diffusion, which we consider in our model (Figure 6). Assignment of Vibrational Peaks upon Solvent Water Isotope Exchange. For RpBphP2, RpBphP3, and its variants, FLN spectra were obtained on samples in protonated and deuterated buffer. Solvent isotope exchange can be used to identify the vibrational modes containing an exchangeable proton site, here, the pyrrole nitrogens. A strong isotope effect is observed, affecting both the overall fluorescence emission band shape and the peak positions of the fine structure. Upon deuteration, the oscillator strength in the FLN spectra vanishes in specific bands (e.g., bands at 1550−1570 cm−1; see Figures 4 and 5), thereby indicating that H/D exchange was efficient. Additionally, a large number of peaks is found mainly in the intervals of 1040−1250 and 1400−1570 cm−1. For the analysis of solvent isotope effects in the FLN spectra, all vibrational peak positions are collected in Table 2; the data for Agp1 and DrBphP variants measured in H2O are also included. Strong intensity vZPL contributions are indicated by a superscript “S”. The wavenumber regions susceptible to deuteration are highlighted in gray. For the phytochrome samples from R. palustris, the proton-associated bands are found at ∼1325 and ∼1570 cm−1, while the deuterium-associated bands are located at ∼1080, ∼1275, ∼1430, ∼1490, and ∼1540 cm−1. The spectra in protonated buffer from phytochromes from A. tumefaciens, D. radiodurans, and R. palustris all display fine structure at similar positions. However, for the variant Agp1PCD-BV15Za, specific bands are found at 903, 996, and 1040 cm−1 that are absent in the FLN spectra of all other BV-binding phytochrome variants. Peaks that evolve strongly or exclusively either in H2O or in D2O buffer for the phytochromes and variants from R. palustris are highlighted by gray boxes in Table 2. At 1570−1573 cm−1, for all variants from R. palustris, a peak is observed in protonated buffer that is not observed in samples prepared in deuterated buffer and thus shows a remarkable isotope effect. This mode is assigned to a N−H vibrational mode. DFT calculations indicate that this frequency is indicative for N−H in-plane bending modes of the two central ring nitrogen atoms of rings B and C.34 Pre-RR spectroscopy revealed a shifting of this band to 1080 cm−1 in deuterated samples. This shift is also clearly observed here by FLN. An intense peak evolves in the FLN spectra in deuterated solvents at a position that is vZPL-silent in protonated solvents. The highest intensity is found at 1078, 1072, 1074, and 1075 cm−1 for the spectra from RpBphP3-PCD and RpBphP3-PCDD216A, which show a good signal-to noise ratio. Two of the

the need for an extended pyrrole water network and the conserved salt bridge between the aspartate side chain and a conserved arginine in the PHY domain28 for phytochrome function. By comparison of the FLN spectra from wild-type and the mutant RpBphP3-PCD-D216A, aspartate is found to provide a coordinate along which proton mobility can occur. Thus, this aspartate is most probably connected directly to the pigment by a hydrogen bond via the backbone carbonyl, consistent with the structural data for RpBphP3.4 Proton Dynamics and Line Narrowing. Upon replacing protonated by deuterated solvent, two major alterations are observed in the FLN spectra of bacteriophytochromes. First, isotope-dependent vibrational peak shifts occur, and second, the number of resolved vZPLs on top of broad fluorescence emission bands increases significantly for all phytochrome variants from R. palustris (Figure 4). As derived from water solvent isotope-dependent single-molecule spectroscopy on PSI, proton dynamics may strongly influence the degree of spectral dynamics of protein-bound pigments at low-temperature conditions of a few Kelvins.73 At such low-temperature conditions, conformational changes are largely frozen out. While protons maintain relatively high mobility at very low temperatures, the higher-mass deuterium isotope is less mobile, and therefore, spectral dynamics seem, in general, to be reduced in deuterated solvents.73 The increased line narrowing efficiency in deuterated solvents is consistent with these observations. While peak shifts indicate efficient isotope exchange at specific chromophore sites, the increased line narrowing efficiency reports a lower degree of spectral diffusion in the case of the deuterated solvent. In bacteriophytochrome, in addition, another mechanism may contribute to the observed deuterated solvent effect on spectral diffusion that relates to the experimental observation that the electronic transition is directly coupled to proton transfer (see the scheme in Figure 6). Ultrafast spectroscopy on RpBphP3 and RpBphP2 has indicated that an ESPT process of the BV chromophore to the protein environment takes place that results in excited-state deactivation and a subsequent rapid back-shuttling of the proton to BV.29,49 On the basis of the increased excited-state lifetime and deuterium isotope effects on the RpBphP3 D216A mutant and the RpBphP3 CBD construct, it was proposed that ESPT from the pyrrole nitrogens of ring A, B, or C to the backbone carbonyl of Asp-216 or to pyrrole water would occur.29 In this way, the pyrrole nitrogen proton/deuteron is actively shuttled between the chromophore and binding pocket by photon absorption, and it thus may sample various conformations, leading to spectral diffusion. Here, the rate of spectral diffusion will depend on the rate of proton/deuteron transfer in the ESPT reaction in wild-type RpBphP3 and RpBphP2, as reported in refs 29 and 49. The enhanced line narrowing in deuterated solvent, in the D216A mutant and in the PCD construct, supports this idea. Phytochrome Variant with Synthesized Pigment. The synthesized tetrapyrrole pigment has an additional covalent bond linking ring C and ring D of the tetrapyrrole chromophore, which abolishes the C15C16 isomerization reaction. When this pigment is bound to the PCD of Agp1 (Agp1-PCD-BV15Za), the variant has strongly altered spectral characteristics compared to wild-type, with well-resolved fine structure and distinct vibrational peak positions in FLN (Table 2). For Agp1-PCD-BV15Za, a temperature-dependent measurement series was taken (Figure 2). The fine structure shows 14947



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promoter for a hydrogen bond network. Strong narrowing of the spectral bands is observed for an Agp1 variant with a synthesized pigment, indicating a pyrrole water network around the chromophore that is less extended or a conformation that does not enable ESPT-coupled spectral diffusion. On the basis of similar FLN characteristics for RpBphP2 and RpBphP3, we infer that a similarly extended hydrogen bond network exists around their chromophores despite the different photoreactions that these phytochromes exhibit at later stages of their photocycles.

four multiline patterns are found in this wavenumber region. The highest-intensity peak occurs in one multiline pattern with interpeak distances of ∼30 cm−1, with one peak at lower and two peaks at higher wavenumbers. On the basis of these observations and the results from DFT calculations,34 we assign the ∼1572 and ∼1074 cm−1 modes to the N−H/N−D in-plane modes of the ring B and C nitrogen atoms, respectively. In the protonated samples from R. palustris for all variants, an intense FLN peak is found between 1324 and 1327 cm−1, which is the same with respect to the experimental resolution. In Pre-RR spectra, this mode cannot be assigned unambiguously because a large number of closely spaced bands in this region also stems from apoprotein modes. As the FLN technique is sensitive solely to the chromophore, the peak at 1324−1327 cm−1 is most probably associated with the N−H inplane bending modes of rings A and D, an association confirmed by DFT calculations.34 In deuterated solvents, the peak at 1327 cm−1 is either not resolved (RpBphP2, RpBphP3) or is part of a multipattern with line spacing of 13−20 cm−1 (RpBphP3-PCD and RpBphP3-PCD-D216A). A remaining contribution of protonated nitrogen sites either is present for these samples or, as this mode is part of a multiline pattern only observable for samples in deuterated solution, is associated with a mode that is frequency-downshifted in deuterated solvents. In the region of ∼1419−1540 cm−1, a further strong isotope effect is observed. In deuterated buffer, an intense, four-line pattern with interpeak distances of ∼15−30 cm−1 contributes to the FLN spectra from RpBphP2, RpBphP3-PCD, and RpBphP3PCD-D216A, but in protonated buffer, this region does not show narrow FLN lines. This spectral region can also be identified as related to the exchangeable nitrogen sites of the tetrapyrrole in its deuterated form. Phytochromes from R. palustris, Agp1, and DrBphP in protonated buffer all show similar vibrational substructure at ∼1320 and ∼1570 cm−1. We assume that equivalent sites are responsible as these phytochromes all bind the same BV pigment. Thus, we attribute peaks at ∼1320 and ∼1570 cm−1 for all protonated phytochromes to modes involving N−H vibrations at rings A/ D and B/C, respectively. One mode that does not show an isotope effect but shows an intense peak contribution in the FLN spectra for all samples is found at ∼800 cm−1. DFT calculations predict a C−H out-ofplane mode in this region of the spectrum.34 As carbon sites do not harbor exchangeable protons at the pH values used, no isotope effect is expected. Thus, this line is in accord with the unchanged peak positions in FLN arising from C−H out-ofplane modes. Likewise, no major isotope effect is observed in the range expected for C−C and CO stretching modes at 1580−1700 cm−1.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Addresses #

J.B.N.: ICFO − Institute of Photonic Sciences, Av. Carl Friedrich Gauss num. 3, 08860 Castelldefels, Barcelona, Spain. § E.A.S.: Department of Biology, Northeastern Illinois University, 5500 N. St. Louis Ave., Chicago, IL 60625, United States. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research was funded by the European Community  Access to Research Infrastructures of the “Improving Human Research Potential” Program, Contract No. RII-CT-2003506350. LaserLaB Europe Program lcvu_1512 “Fluorescence line narrowing on phytochromes” and measurements conducted at the LaserLaB Europe Vrije Universiteit Amsterdam. J.T.M.K. was supported by the Chemical Sciences Council of The Netherlands Foundation for Scientific Research through a VICI grant. K.M. and E.S. were supported by NIH Grant GM036452 to K.M. We thank André Verméglio, CEA Cadarache for wild-type phytochrome samples from R. palustris.

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ABBREVIATIONS A. tumefaciens, Agrobacterium tumefaciens; R. palustris, Rhodopseudomonas palustris; D. radiodurans, Deinococcus radiodurans; CBD, chromophore binding domain (PAS-GAF); FLN, fluorescence line narrowing spectroscopy; PCD, photosensory core domain (PAS-GAF-PHY); ESPT, excited-state proton transfer

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REFERENCES

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CONCLUSION The different widths of spectral lines observed in the FLN spectra for phytochromes from different species indicate different proton mobility in their chromophore binding pockets. By solvent isotope exchange, vibrational modes involving exchangeable proton sites that contribute to the FLN spectra were identified. Line narrowing is increased in deuterated compared to protonated solvents, consistent with induction of spectral line broadening by proton dynamics, which is possibly related to the ESPT reactions observed previously in bacteriophytochrome.29,49 On the basis of analysis of a point mutant of RpBphP3, an aspartate residue in the direct vicinity of the chromophore is identified as being a 14948



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