Fig 1.
Homological models and amino acid sequences of POR proteins.
(A) Aligned homological models of PORA (blue), PORB (yellow) and PORC (green). Structures of pairs of proteins (PORA/PORB, PORA/PORC and PORB/PORC) are overlaid. (B) Aligned sequences of PORC, PORA and PORB. Identical residues are highlighted in black. Red line marks transit peptide cleavage site. Some mismatches in the structure alignments obtained between PORA and PORC are marked in the orange frame, while those between PORA and PORB—in the cyan one. Characteristic motifs are marked with red letters, namely the G-rich motif, the NAA motif, the TFT motif and the catalytic YxxxK motif.
Fig 2.
Fluorescence studies of reaction mixtures containing PORA, Pchlide, and NADPH (A) Representative 77 K fluorescence emission spectra; the Pchlide:PORA ratio is shown in the legend.
Excitation wavelength: 440 nm. (B) The relation between the position of the 77 K fluorescence emission maximum of Pchlide (determined on the basis of spectra similar to those shown in Fig. 2A) and the Pchlide:POR ratio in reaction mixtures containing Pchlide, NADPH and either PORA, PORB or PORC, respectively. The presented results were obtained from several experiments performed for different POR concentration between 4 and 11 μM, and therefore were analysed with respect to Pchlide:POR ratio.
Fig 3.
Representative 77 K fluorescence emission spectra of a reaction mixture containing Pchlide, NADPH and PORA or PORC.
Spectra labelled as “dark” were measured after a 30-min incubation of the reaction mixture in darkness. After these measurements, the samples were thawed, illuminated, frozen again and used for fluorescence measurement (spectra labelled as “light”). See materials and methods for the details. POR concentration: 6.3 ± 0.3 μM, Pchlide concentration: 1.3 μM, NADPH concentration: 0.2 mM. Pchlide:POR ratio = 0.21. Excitation wavelength: 440 nm.
Fig 4.
The dependence of the relative Chlide fluorescence intensity on the Pchlide:POR ratio.
The relative Chlide fluorescence intensity was calculated as FChlide/(FChlide+FPchlide). Fluorescence intensity of Chlide (FChlide) and Pchlide (FPchlide) were read from 77 K fluorescence emission spectra, around 680 and 640 nm, respectively, that were measured for reaction mixtures after 15 sec of illumination with white light (8 μmol m-2 s-1 photon flux density). Excitation wavelength: 440 nm. The presented results were obtained from several experiments performed for different POR concentration between 4 and 11 μM, and therefore were analysed with respect to Pchlide:POR ratio.
Fig 5.
The dependence of the maximum of the photoactive Pchlide-POR-NADPH complexes on the Pchlide:POR ratio.
Maxima were read from the “dark”-“light” difference spectra obtained for substraction of similar spectra to those shown in Fig. 3 but measured for all the investigated Pchlide:POR ratios. The presented results were obtained from several experiments performed for different POR concentration between 4 and 11 μM, and therefore were analysed with respect to Pchlide:POR ratio.
Fig 6.
Detection of free Pchlide in buffers.
(A) 77 K fluorescence emission spectra of Pchlide in a water-based buffer containing imidazole. Excitation wavelength: 440nm. Pchlide concentration 0.26 μM. (B) 77 K fluorescence excitation spectra for Pchlide in a water-based buffer containing imidazole in the absence and presence of PORA. NADPH concentration: 0.2 mM. Emission wavelength: 660 nm. Pchlide concentration 0.26 μM; log[IMI:Pchlide] = 5.76.
Fig 7.
The dependence of the fluorescence of unbound Pchlide (FunPch) on the Pchlide:POR ratio (A) and on the fluorescence emission maximum (B). FunPch was calculated according to formula (Eq. 1) based on the 77 K fluorescence excitation spectra measured for emission at 660 nm.
The presented results were obtained from several experiments performed for different POR concentration between 4 and 11 μM, and therefore were analysed with respect to Pchlide:POR ratio.
Fig 8.
Relative activity of the POR proteins, defined as the increase in Chlide fluorescence intensity per time unit and per protein concentration (in mg), calculated for different Pchlide concentrations.
Calculations were performed for series of fluorescence spectra measured at room temperature (e.g. S2 Fig.). POR concentration in a single experiment was between 1 and 2.75 μM; NADPH concentration: 0.05 mM. Error bars are SD of two independent series of fluorescence spectra.