Osmolytes Modulate Photoactivation of Phytochrome: Probing Protein Hydration

Abstract

Phytochromes are bistable red/far-red light-responsive photoreceptor proteins found in plants, fungi, and bacteria. Light-activation of the prototypical phytochrome Cph1 from the cyanobacterium Synechocystis sp. PCC 6803 allows photoisomerization of the bilin chromophore in the photosensory module and a subsequent series of intermediate states leading from the red absorbing Pr to the far-red-absorbing Pfr state. We show here via osmotic and hydrostatic pressure-based measurements that hydration of the photoreceptor modulates the photoconversion kinetics in a controlled manner. While small osmolytes like sucrose accelerate Pfr formation, large polymer osmolytes like PEG 4000 delay the formation of Pfr. Thus, we hypothesize that an influx of mobile water into the photosensory domain is necessary for proceeding to the Pfr state. We suggest that protein hydration changes are a molecular event that occurs during photoconversion to Pfr, in addition to light activation, ultrafast electric field changes, photoisomerization, proton release and uptake, and the major conformational change leading to signal transmission, or simultaneously with one of these events. Moreover, we discuss this finding in light of the use of Cph1-PGP as a hydration sensor, e.g., for the characterization of novel hydrogel biomaterials.

Keywords: Cph1; Pfr; Pr; hydration; osmotic stress; phytochrome.

Figure 1

(a) Structural model of Cph1-PGP (PDB: 2VEA). PAS (P, blue), GAF (G, light magenta), and PHY (P, green) denote the domains in the photosensory module (PGP); the amino acid numbers are given in the scheme below together with the output module for signal transduction (yellow). The PCB chromophore is bound to cysteine in position 256. The structural motif, called the tongue, connects the PAS domain with the chromophore binding pocket and changes its conformation between the β-sheet in Pr and the α-helix in Pfr. (b) Cph1 photocycle with red (r) and far red (fr) light excitation. Upon Pr excitation photoisomerization (Pr to Lumi R), chromophore relaxation, transient deprotonation, and large conformational changes (Meta Ra to Meta Rc to Pfr) take place. (c) Potential osmotic stress effects on the Pr-to-Pfr photoreaction are schematically shown. Illustrated is a situation where water changes (water influx) play a role in photoconversion. The photoreaction is shifted either forward or backward, depending on the osmolyte size. Small osmolytes penetrate the protein, thus favoring Pfr formation. Large polymer osmolytes like PEG 4000 are excluded from the protein to withdraw water, thus disfavoring Pfr formation.

Figure 2

Photoconversion kinetics of Cph1-PGP. (a) Absorbance spectra after far-red irradiation (black) and red irradiation (red). (b) Photoconversion kinetics after 20 s of LED irradiation with λ_LED = 680 nm. The time trace can be fitted with a single exponential, allowing approximation by the initial slope method as shown in (c). The time constant of the single exponential fit is given. (c) Determination of the photoconversion rate constant using the initial slope of the –logΔP vs. t curves with ΔP = ([Pfr]_t − [Pfr]_∞)/([Pfr]₀ − [Pfr]_∞). Conditions: 300 mM NaCl, 50 mM Tris, pH 7.8, RT (23 ± 1 °C).

Figure 3

Osmolytes and macromolecular crowding effects on Cph1-PGP photoconversion kinetics. (a) Different osmolytes affect Pr to Pfr kinetics; low molecular weight PEG (35 w% PEG 600) and 35 w% sucrose slightly accelerate photoconversion kinetics, while large PEGs (35 w% PEG 1000, 35 w% PEG 4000) slow down photocycle kinetics. (b) Increased viscosity is not the cause of delayed kinetics, as shown by the comparison of sucrose and PEG 1000 at similar viscosities of 3.9 mPas. For comparison with the effect of a macromolecular crowder, the time trace in a polymeric sucrose (Ficoll 400) solution is also shown. Gray indicates the time trace in buffer.

Figure 4

Estimation of the number of water molecules taken up or released from red light-irradiated Cph1-PGP for polymer (PEG) osmolytes and sucrose according to Equation (3). (a) ln K plotted versus osmotic pressure for different sizes of PEG osmolytes, sucrose, and buffer based on results in Figure 3. The dashed lines guide the eye. For PEG 1000, the values for the different concentrations follow the dashed line, indicating the same apparent change in water molecules based on the slope according to Equation (3). (b) Apparent number (N_W) of water molecules entering or leaving the Cph1 protein interior based on Equation (3).

Figure 5

Photoconversion kinetics of Cph1-PGP in the peptide hydrogel hFF03. (a) Photoconversion kinetics in hFF03 and in buffer (see Figure 2b,c). (b) Absorbance spectra after far-red irradiation (black) and kinetics recording (red). (c) Rate determination from the initial slope. The photoconversion rate is given in the figure. Conditions as in Figure 2 and Table 1.