Figure 1.
Domain architecture and reaction scheme of Cph1.
(A). Domain architecture of phytochrome, consisting of an N-terminal photosensory module that comprises the PAS, GAF and PHY domains, and a C-terminal regulatory module that contains a histidine-kinase like domain (HK) [1]. The Cys residues that have been used for spin-labeling studies in the present work are shown and the green asterisk indicates the phycocyanobilin cofactor. (B). The proposed photoconversion of the Pr to Pfr states of the phycocyanobilin cofactor in Cph1 phytochrome. The chromophore in the Pr state is shown as ZZZssa at the AB, BC, and CD rings, respectively [5]. Illumination with red light triggers photoisomerization about the C15–C16 methine bridge to give the primary photoproduct, Lumi-R, which is subsequently converted to Pfr in several light-independent steps on the millisecond-to-second timescale. The Pr state can be regenerated from Pfr by excitation with far-red light or by a slow dark reversion process [1].
Figure 2.
PELDOR analysis of Cph1 conformations.
PELDOR data obtained from spin-labeled Synechocystis PCC 6803 Cph1. Raw four-pulse ELDOR traces together with the third order polynomial function used to baseline the data (in red) are shown in the left hand panel, while the right hand panel shows the conjugate Fourier transforms of these data. (A) and (G). Pr form of the N-terminal photosensory region with spin-label at C371. (B) and (H). Pfr form of the N-terminal photosensory region with spin-label at C371. (C) and (I). Pr form of full-length Cph1 with spin-label at C371. (D) and (J). Pfr form of full-length Cph1 with spin-label at C371. (E) and (K). Pr form of full-length Cph1 with spin-label at C371 and N733C. (F) and (L). Pfr form of full-length Cph1 with spin-label at C371 and N733C. Pulse sequences and data processing are described in Materials and Methods. Numbers in the right hand panel indicate νDD and distances referred to in the text.
Figure 3.
Characterization of the Pr → Pfr conversion by cryogenic absorbance measurements.
(A). 77 K absorbance difference spectra of samples containing 15 µM Cph1 after illumination for 10 mins at different temperatures ranging from 77 K to 180 K. The difference spectra were obtained by using a non-illuminated sample as a blank. The formation of the absorbance peak at 684 nm and simultaneous disappearance of the absorbance band at 668 nm at higher temperatures are indicated by the arrows. (B). 77 K absorbance difference spectra of samples containing 15 µM Cph1 after illumination at 180 K for 10 mins and incubation in the dark for 10 mins at increasing temperatures. The difference spectra were obtained by using the sample that was illuminated at 180 K as a blank. The arrows indicate the formation and disappearance of the different absorbance bands at higher temperatures. The raw absorbance spectra are shown in figure S4.
Figure 4.
The temperature dependence of the steps involved in the Pr → Pfr photoconversion.
The temperature dependence of the initial formation of the Lumi-R state (•) and the remaining step(s) to form the Pfr state (○) were obtained by analysis of the cryogenic absorbance measurements (Figure S5) and are shown together with the kT value for each step, where k is the Boltzmann constant (0.695 cm−1) and T is the mid-point temperature of each process. The ‘glass transition’ temperature of proteins is also shown as a reference (dashed line).
Figure 5.
Characterization of the initial photoisomerization dynamics of the Pr state of Cph1.
Cph1 was contained in a 5% sucrose solution and absorbance spectra recorded after photoexcitation with a laser pulse centred at ∼590 nm. (A) Transient absorption difference spectra at delay times of 1, 5, 10, 29, 60, 299, and 988 ps after excitation. (B) Kinetic transient at 673 nm (black squares) with a fit of the data to 2 exponentials shown in red.
Table 1.
The effect of solvent viscosity (η) on the lifetimes associated with the initial photoisomerization dynamics of the Pr state of Cph1.
Figure 6.
Characterization of the Pr → Pfr conversion by laser photoexcitaion measurements.
(A) Absorbance difference (‘action’) spectra following photoexcitation of 15 µM Cph1 (Pr form) with a 6 ns laser pulse at 660 nm. The spectra were created by measuring the absorbance change at the respective wavelength at various timepoints after laser excitation. (B) Typical kinetic traces measured at 720 nm over 1 second on a logarithmic timescale following photoexcitation of 15 µM Cph1 (Pr form) with a 6 ns laser pulse at 660 nm. The data were fitted to 4 kinetic phases to obtain rate constants (red line). The inset shows kinetic transients at 720 nm at 0% sucrose and 30% sucrose to illustrate the effect of solvent viscosity. All traces were collected at 20°C as described in the Materials and Methods section.
Table 2.
The effect of solvent on the rates of the slower steps in the Pr → Pfr photoconversion.
Figure 7.
The viscosity-dependence of the slower steps in the Pr → Pfr photoconversion.
The viscosity dependence of the rate constant for the 1st (A), 2nd (B), 3rd (C) and 4th steps (D) of the increase in absorbance at 720 nm are shown. All measurements were recorded over a range of timescales and the data are fitted to equation 2 as described in the Materials and Methods. The error bars were calculated from the average of at least 3 traces.
Figure 8.
Scheme showing proposed domain movements during photoconversion of the Pr to Pfr forms of Cph1.
The asterisks indicate the phycocyanobilin cofactor.