Revealing the origin of multiphasic dynamic behaviors in cyanobacteriochrome

Significance

Cyanobacteriochromes detect a broad range of light conditions to regulate various physiological processes in cyanobacteria. However, the initial photoinduced isomerization reaction in cyanobacteriochromes has not been well understood. The coupling motion of the excited chromophore with the local protein environment has not been carefully addressed and the often-observed multiphasic dynamic behavior was mostly attributed to the ground-state heterogeneity. Here, by integrating both femtosecond-resolved fluorescence and absorption methods, we revealed significant active-site solvation and thus elucidate a new molecular picture of photoisomerization, paving the way to explain many observations in cyanobacteriochromes and phytochromes.

Abstract

Cyanobacteriochromes are photoreceptors in cyanobacteria that exhibit a wide spectral coverage and unique photophysical properties from the photoinduced isomerization of a linear tetrapyrrole chromophore. Here, we integrate femtosecond-resolved fluorescence and transient-absorption methods and unambiguously showed the significant solvation dynamics occurring at the active site from a few to hundreds of picoseconds. These motions of local water molecules and polar side chains are continuously convoluted with the isomerization reaction, leading to a nonequilibrium processes with continuous active-site motions. By mutations of critical residues at the active site, the modified local structures become looser, resulting in faster solvation relaxations and isomerization reaction. The observation of solvation dynamics is significant and critical to the correct interpretation of often-observed multiphasic dynamic behaviors, and thus the previously invoked ground-state heterogeneity may not be relevant to the excited-state isomerization reaction.

Cyanobacteriochromes (CBCRs) are bilin-based photoreceptors in cyanobacteria that regulate various physiological functions including biofilm formation, complementary chromatic adaptation, and phototaxis (1–4). The light-sensing GAF domain covalently binds to a phycocyanobilin (PCB) chromophore and usually occurs in tandem with other sensory domains and thus multiple environmental signals are integrated to finely tune such diverse biological activities (Fig. 1A) (5). Light signal triggers the GAF domain conformational changes through the photoinduced E/Z isomerization about the C₁₅=C₁₆ double bond of the chromophore (Fig. 1B) (5). CBCRs are classified into several subfamilies that exhibit extremely diverse spectral properties covering from near-UV to far-red region by two photoconvertible states (6–10). Unlike classic phytochromes with far-red light sensitivity, red/green CBCRs photoconvert between a green-absorbing 15E state (Pg) and a red-absorbing 15Z state (Pr). The spectral tuning and photoisomerization mechanism of such unique blue-shifted state has been recently studied by transient-absorption methods (11–14). However, the origins of multiphasic decays of isomerization dynamics, ranging from subpicosecond to hundreds-of-picoseconds timescale, are still under debate, and the effects of protein active-site relaxation upon sudden excitation on ultrafast photoisomerization dynamics have not been addressed yet.

Fig. 1.

(A) Surface and ribbon representations of Pg-state PPHK dimer. The nREC domain (red) is connected to the GAF domain (blue) through a long helical structure commonly seen in the phytochrome family. The chromophore PCB (purple) is covalently linked to the GAF domain and exposed to the solvent. (B) Close-up view of the local chromophore binding pocket with water molecules found in the crystal structure (Protein Data Bank ID code 6OAP). The chromophore is in close contact with the negatively charged D211 (yellow) and E210 (green). The Phe cluster of F180, F214, and F249 (green) is also shown. Q228 (green) has polar contacts to the chromophore propionate side chains.

To resolve these important issues, we systematically studied the excited Pg-state isomerization dynamics in a recently discovered CBCR, PPHK (phosphorylation-responsive photosensitive histidine kinase) tandem sensor domains from cyanobacterium Leptolyngbya sp. JSC-1 (Fig. 1) (15, 16). The phosphorylation-sensing N-terminal receiver (nREC) domain is connected to the light-sensing GAF domain by the long helical structure (17, 18). The GAF domain alone covalently incorporates the chromophore through a conserved cysteine residue (C241), and the shallow active site is readily accessible to the solvent (Fig. 1). The green-absorbing Pg state in PPHK is modulated by a unique phenylalanine (Phe) cluster, including the β1-Phe (F180), the Asp-motif Phe (F214), and the helix-Phe (F249) (Fig. 1B and SI Appendix, Fig. S1A) (15). The PCB chromophore has tight hydrogen-bonding interactions with the aspartic acid residue (D211) of the conserved Asp-motif. The B- and C-ring propionate groups are deeply buried in the GAF domain surrounded with extensive polar contacts. Here, we report our systematic characterization of the Pg-state photoisomerization dynamics in wild-type (WT) PPHK and five critical mutants (E210L, Q228L, F180L, F214L, and F249L) using both femtosecond-resolved fluorescence and absorption methods. By following the femtosecond-resolved fluorescence evolution of the excited-state chromophore, we observed the solvation dynamics of the active site and obtained the actual relaxation times. We further studied the isomerization dynamics of reactants, various intermediates and photoproducts with a wide absorption probing wavelength from 350 to 720 nm. We finally elucidate the Pg-state isomerization mechanism and reveal the critical role of the protein environment on the ultrafast Pg-state reaction.

Results and Discussion

Femtosecond Fluorescence Dynamics and Active-Site Solvation.

Fig. 2A shows the steady-state absorption and emission spectra of WT, E210L, Q228L, F214L, and F249L in the Pg state. The WT absorption spectrum has its peak at 564 nm. The emission spectrum peaking at 640 nm is approximately the mirror image of the absorption spectrum. All of the mutants show similar absorption and emission peaks (SI Appendix, Table S1). The obvious broadening of the absorption spectra of F214L and F249L results from spectral tuning of the surrounding Phe cluster at the active site. Importantly, we here observed a large Stokes shift in both WT and the mutants.

Fig. 2.

(A) The Pg steady-state absorption and emission spectra of WT (red), E210L (blue), F214L (dark cyan), Q228L (yellow), and F249L (orange). The pump wavelength at 560 nm and multiple probe wavelengths (colored downward arrows) for the transient absorption detection as well as gated fluorescence wavelengths (black upward arrows) are marked. (B) Normalized femtosecond-resolved fluorescence transients of Pg-state WT PPHK gated at typical wavelengths in the linear (before 20 ps) and logarithmic timescales. All of the experimental data are shown in circles, and the solid lines are the best exponential fit. (C and D) Normalized femtosecond-resolved fluorescence transients of mutants as comparison with WT gated at 620 nm (C) and 700 nm (D). Note that the delay time is shown on a linear scale before 40 ps and a logarithmic scale thereafter.

Fig. 2B shows the femtosecond-resolved WT fluorescence transients at several typical wavelengths gated from the blue to red side of the emission within a 3.2-ns time window. Besides a lifetime component of 730 ps in each transient, we observed decays at the blue side and rises at the red side, a typical manifestation of the local solvation dynamics (SI Appendix, Table S2) (19–21). The transients at the blue side are fitted with three solvation components ranging from 0.8 to 3.5, 15 to 31, and 150 to 250 ps. At the red side, the transients show two rise components in 1.2 to 3.5 and 20 to 31 ps. These dynamics are from the active-site relaxation of polar side chains and water molecules, as observed in photolyases, flavodoxin, and GFP variants (22–27). The relatively long excited-state lifetime (730 ps) of the Pg state enables the characterization of continuous solvation dynamics from a few to tens and to hundreds of picoseconds in PPHK. All three components are obviously present, and thus the solvation dynamics is significant. Such direct observation of active-site solvation dynamics in CBCR is crucial to the understanding of the excited-state dynamics, especially with transient-absorption detection below. We also measured the femtosecond-resolved fluorescence anisotropy dynamics of the chromophore at 660-nm emission (SI Appendix, Fig. S2B). Surprisingly, we did not observe any decay dynamics up to a few hundreds of picoseconds, indicating a relatively immobile excited-state chromophore confined at the active site electrostatically and structurally. The observed solvation dynamics are mainly from the local water and protein side-chain relaxations.

Fig. 2 C and D show the femtosecond-resolved fluorescence transients of mutants with WT as comparison for two typical wavelengths at 620 nm (blue side) and 700 nm (red side) out of a series of wavelengths detection (SI Appendix, Figs. S3–S7). The observed solvation processes at the blue side for the mutants are all faster than those for WT, while the lifetime components vary among mutants (Fig. 2D and SI Appendix, Tables S2–S6). For example, for the mutant E210L, the solvation dynamics at the blue side occur in 1.2 to 3.2, 11 to 29, and 85 to 160 ps and rise in 1.2 to 3.0 and 17 to 26 ps at the red side with a shorter lifetime of 550 ps. For Q228L, the solvation relaxations are in 1.5 to 3, 18 to 37, and 160 to 260 ps, while at the red side they show rises in 1.1 to 3.0 and 19 to 30 ps. These faster solvation dynamics in the mutants are closely correlated with local active-site properties, as discussed below.

To extract solvation correlation function, we directly measured the femtosecond-resolved emission spectra (FRES). Fig. 3A displays a three-dimensional (3D) map of fluorescence emission relative to emission wavelength and delay time, and Fig. 3B shows several snapshots at various delay times for the WT PPHK. We observed obvious dynamic Stokes shifts on a timescale of a few to hundreds of picoseconds. At 0.5 ps, the spectrum peaks around 631 nm and at a later time of 1 ns, the peak moves to 640 nm. To quantify the local relaxation dynamics, we calculated the averaged frequencies ($\overline{\nu }$) and their changes with time to represent the solvation dynamics (see SI Appendix and refs. 22 and 23). We observed a total shift of 194 cm⁻¹ for the WT and the normalized correlation function is shown in Fig. 3C. The solvation dynamics was observed to occur in 2.5 (15%), 25 (47%), and 235 ps (38%). Upon photoexcitation, the active site continues to evolve in dynamic motions from a few picoseconds until isomerization in several hundreds of picoseconds. The X-ray structure of PPHK shows about six trapped water molecules within 8 Å of the chromophore center (Fig. 1B) (16). The reported 10-ns molecular-dynamics (MD) simulation of bacteriophytochrome DrBphP found extensive water exchanges between the chromophore region and the outside solvent, resulting in continuous electrostatic fluctuations in the GAF domain (28). Therefore, significant water–protein dynamic interactions for an opened active site are expected in PPHK. Thus, the first solvation component of 2.5 ps mainly reflects the local water-network relaxation at the active site. The observed relaxation dynamics in tens and hundreds of picoseconds represent the collective rearrangements of local water molecules and their interacting side chains. Moreover, the PCB chromophore is tightly “clamped” by the GAF domain as observed above; thus, such observed dynamic motions continuously tune the excited-state potential surface as well as the local side-chain configurations to finally facilitate the D-ring flipping, which in turn initiates further protein structural changes.

Fig. 3.

(A) A 3D representation of femtosecond-resolved emission spectra (FRES) of WT PPHK. The intensity is scaled by a color code. (B) Snapshots of the FRES at several typical delay times. The steady-state emission peak is also shown with the dotted line. (C) Solvation correlation functions for the Pg-state PPHK WT and two mutants of E210L and Q228L. The dashed lines show each exponential decay components (τ_1S, τ_2S, and τ_3S).

Critical Site Mutations and Modulation of Dynamic Evolution.

The 15E Pg-state reported here exhibits an extended excited-state lifetime compared to the ultrafast 15E Pfr-state dynamics of a few picoseconds in the phytochromes (29), primarily due to the effects of the unique protein environment. With such a long lifetime, the isomerization dynamics mixes with the local environment relaxations. Here, we examine the effect of mutations at the active site on relaxation and isomerization dynamics. We studied the solvation dynamics of two mutants E210L and Q228L in detail, and the results are given in Fig. 3C and Table 1. Both solvation dynamics of the two mutants become obviously faster. Substituting E210 near the positively charged PCB chromophore has significant influence on the water network between the Asp-motif and the chromophore. For E210L, we observed the solvation dynamics in 1.6 (34%), 68 (37%), and 151 ps (29%) with a total stabilization energy of 200 cm⁻¹, and all of the relaxation times are faster than those of WT. The observed fast solvation dynamics indicate that the local water network and protein side chains become more flexible when removing the charged E211 near the Asp-motif. The PCB chromophore is anchored in the GAF domain by the hydrogen-bonding interactions with the Asp-motif. The modification by mutation of E210L near the structurally conserved Asp-motif disturbs such local hydrogen-bonding networks, resulting in fast collective water–protein motions. Overall, the fast solvation dynamics and accelerated excited-state lifetime show that the Asp-motif interactions are critical to the excited-state photoisomerization dynamics in PPHK.

Table 1.

Results of solvation dynamics of PPHK WT and mutants

	τ1	ΔE1	τ2	ΔE2	τ3	ΔE3	ΔET
WT	2.5	33	25	89	235	72	194
E210L	1.6	68	18	74	151	58	200
Q228L	2.3	58	26	84	231	73	215

Time constant (τ) and stabilization energy (ΔE) are in units of picoseconds and centimeter⁻¹, respectively.

The mutation of Q228L also leads to faster relaxations. The solvation correlation function shows the dynamics in 2.3 (27%), 26 (39%), and 231 ps (34%) with a large stabilization energy of 215 cm⁻¹. In Pg state, the Q228 is within hydrogen-bonding distances with both the B- and C-ring propionate group, which is part of extensive water-mediated hydrogen-bonding networks among the chromophore side groups with nearby polar residues (Fig. 1B). Due to the replacement of glutamine by leucine, the hydrogen-bonding networks are partially broken, causing the local structure to be relatively flexible, but the rotation of PCB chromophore relative to the protein takes place at a much later stage of isomerization (16). We did not observe the change of excited-state lifetime because the mutation of Q228L does not directly affect the D-ring environment, as observed in bacteriophytochrome PaBphP; the Pfr (15E) excited-state lifetime remains nearly unchanged by a similar mutation of S275A in PaBphP (29). Thus, the elimination of PCB propionate hydrogen-bonding interactions results in more flexible local hydrogen-bonding networks and protein side-chain motions.

Similarly, the Phe mutations of F180L, F214L, and F249L cause the active site to be flexible and result in faster relaxation. The chromophore D-ring is sandwiched between the β1 F180 and the helix F249 (SI Appendix, Fig. S1A). During the photoisomerization, the D-ring rotates and has to push away the aromatic rings in a diffusive manner (30). The structural perturbation at these two sites near the D-ring as well as Asp-motif F214 also accelerates the photoisomerization dynamics, leading to a shorter lifetime of 570 ps for F180L, 635 ps for F249L, and 604 ps for F214L. Again, the mutations of active-site residues near the D-ring all cause a loose local structure, resulting in faster solvation relaxations and isomerization.

With mutations, we have observed the faster active-site solvation and altered isomerization dynamics, indicating the collective contributions of the active site, chemically and structurally. The ultrafast structural dynamics of phytochrome in a few picoseconds after photoexcitation, including the motions of side chains as well as active-site water, has been reported recently by Claesson et al. (31). Thus, the initial ultrafast structural response collectively occurs in a few picoseconds and the correlated rearrangements of water molecules and protein side chains happen in a longer timescale in tens and hundreds of picoseconds, followed by the isomerization to trigger large-conformation changes leading to function. In CBCR, the Phe cluster as well as the Asp-motif region are involved in such allosteric structural events. More theoretical/computational work is needed for the complete understanding of such ultrafast nonequilibrium dynamics in terms of structural changes.

Isomerization Reaction and Dynamic Heterogeneity.

To fully characterize Pg-state isomerization reaction, we switched to the transient-absorption detection (Fig. 4 and SI Appendix, Figs. S7–S9). Specifically, at 350 nm, we detected ground-state recovery in hundreds of picoseconds and photoproduct formation. The positive plateau attributes to the absorption of the photoproduct Lumi-G. Transient signals probed at 440, 680, and 720 nm mainly reflect the excited-state dynamics. Transient signals probed at 600, 620, and 650 nm are complex and consist of ground-state recovery, stimulated emission, and product formation. Solvation dynamics can be readily observed in these transient-absorption measurements (26, 32, 33). The transient-absorption data of WT and the mutants are fitted with sum of various exponential decays, and the fitting results are listed in SI Appendix, Tables S7–S10. Besides the excited-state lifetime components, three time constants of about 1.2 to 4.8, 19 to 38, and 91 to 285 ps were observed, and they are from the local structural relaxations of solvation processes. In many previous femtosecond-resolved transient absorption studies of phytochrome/CBCR photoisomerization dynamics (12, 13, 34), the multiple exponential decays are often assigned to the dynamics of heterogeneous ground-state subpopulations. Here, by resolving the solvation dynamics, we have clearly shown that these multiphasic decays result from the local environment relaxations, not from a ground-state heterogeneity (35, 36). Thus, combining both femtosecond-resolved fluorescence and absorption detections is crucial to the correct interpretation of excited-state isomerization. Integrating both fluorescence and absorption results together, we paint the molecular mechanism of isomerization reaction along two coordinates, nuclear and solvation (Fig. 5). The reaction initially occurs along solvation coordinate, representing the active-site solvation and the local trapped water and polar side-chain relaxations in a few to hundreds of picoseconds. Then, the reaction moves to nuclear coordinate to proceed to isomerization. Such the double-bond twisting is a nonequilibrium process, leading to continuous local relaxations and resulting in the reaction to continuously evolve along both solvation and nuclear coordinates as shown in Fig. 5.

Fig. 4.

Normalized femtosecond-resolved absorption transients of WT PPHK probed from 350 to 720 nm. Top Inset shows the gradual changes of the dynamics with different probe wavelengths. Middle and Bottom Insets show the deconvolution of the transients into various dynamic components probed at 650 nm on short and long timescales. All of the experimental data are shown in circles, and the solid lines are the best exponential fit.

Fig. 5.

Schematic potential energy surface for PPHK Pg-state photoisomerization along the solvation and reaction coordinates. The excited state (Pg) evolves first along solvation coordinate in a few picoseconds to hundreds of picoseconds. The isomerization, convoluted with continuous local motions, passes a barrier and decays to the ground state through a conical intersection (CI) to partially form Lumi-G photoproduct.

Conclusion

We reported here our systematic characterization of the solvation dynamics and isomerization reaction of the Pg state in PPHK. With the femtosecond-resolved fluorescence method, we obtained the complete solvation picture and observed the active-site relaxation process in PPHK occurring from a few to hundreds of picoseconds, revealing a continuous structural evolution after photoexcitation. This observation is critical and resolves the long debating about the origin of the excited-state multiple exponential decays, especially observed extensively by the transient-absorption methods in literature. The isomerization evolves along both solvation and nuclear coordinates, a nonequilibrium process involving local water molecules and polar side chains in constant relaxations (Fig. 5). Our transient-absorption detection fully confirmed the above picture, and the observed dynamics are mixed with isomerization reaction and solvation processes. We also studied several critical mutants at the active site, and these modifications by mutations lead to looser local structures, resulting in faster local relaxations and subsequent isomerization reactions. Finally, the complete separation of active-site solvation and excited-state isomerization is critical to a correct interpretation of the entire dynamic evolution and an accurate description of the reaction mechanism. The local environment relaxations must be included in the understanding of the photoisomerization dynamics. These results are no doubt significant and can be generalized to other CBCRs and phytochromes.

Materials and Methods

Protein Preparation.

The tandem sensor domains of WT and five mutants of the cyanobacterium PPHK were overexpressed in Escherichia coli BL21 (DE3). The protein expression and purification followed procedures reported previously (16). The steady-state fluorescence emission was measured using a SPEX FluoroMax-3 spectrometer with sample concentration at 5 to 10 μM. For the femtosecond-resolved experiments, the PPHK samples were prepared in a 50 mM NaCl and 20 mM Tris buffer (pH 8) with a sample concentration of 50 to 200 μM. During the experiment, the samples were under constant illumination of a 660-nm peak output of LED light for complete conversion of Pr state to Pg state for femtosecond studies.

Femtosecond Methods.

Femtosecond-resolved measurements were taken using the fluorescence upconversion and transient absorption methods. The experimental layout has been described previously (37–39). Briefly, for the fluorescence upconversion experiments, the pump pulse was set at 560 nm with the energy attenuated to ∼100 nJ before it was focused into the sample cell. The fluorescence emission signals were gated by another 800-nm laser beam in a 0.5-mm-thick β-barium borate crystal (BBO, type I). For the transient absorption experiments, the probe pulses at the desired wavelengths between 350 and 720 nm were generated via optical parametric amplifiers (TOPAS; Spectra-Physics). The instrument responses are 400 fs and 120 to 200 fs for fluorescence and transient absorption detection, respectively. All experiments were conducted at the magic angle (54.7°). For fluorescence anisotropy measurements, the polarization of the pump beam was adjusted to be either parallel or perpendicular to the acceptance axis to obtain the parallel and perpendicular signals. To prevent heating and photobleaching, the sample was kept in stirring quartz cells with a 1- or 5-mm thickness during laser irradiation. The femtosecond-resolved data were fitting by multiple exponential decays. More data analyses are described in SI Appendix.

Data Availability.

All data relevant to this research are available in the main text and SI Appendix.