Plant LHC-like proteins show robust folding and static non-photochemical quenching

Abstract

Life on Earth depends on photosynthesis, the conversion of light energy into chemical energy. Plants collect photons by light harvesting complexes (LHC)-abundant membrane proteins containing chlorophyll and xanthophyll molecules. LHC-like proteins are similar in their amino acid sequence to true LHC antennae, however, they rather serve a photoprotective function. Whether the LHC-like proteins bind pigments has remained unclear. Here, we characterize plant LHC-like proteins (LIL3 and ELIP2) produced in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Both proteins were associated with chlorophyll a (Chl) and zeaxanthin and LIL3 was shown to be capable of quenching Chl fluorescence via direct energy transfer from the Chl Q_y state to zeaxanthin S₁ state. Interestingly, the ability of the ELIP2 protein to quench can be acquired by modifying its N-terminal sequence. By employing Synechocystis carotenoid mutants and site-directed mutagenesis we demonstrate that, although LIL3 does not need pigments for folding, pigments stabilize the LIL3 dimer.

Fig. 1. Isolation and analysis of LIL3 and ELIP2 proteins.

a A scheme of LIL3 and ELIP2 proteins; the Chl-binding ‘Hlip’ helix is drawn as an orange rectangle and the ExxNxR motif is highlighted as a red strip. b 2D CN/SDS-PAGE of the purified (pur.) LIL3 and ELIP2 proteins after sucrose gradient (Supplementary Fig. 2); 1.4 μg of Chl was loaded for each sample. The native gel was scanned (Scan) and Chl fluorescence (Ch FL) was detected after excitation with blue light. Separated Synechocystis membrane complexes (5 μg of Chl) are shown as a ‘mass’ control: PSI[3]—trimeric PSI (1 MDa), PSI[1] monomeric PSI (300 KDa), Cpc[6]—a hexamer of phycobilisome rod subunits, (100 KDa). LIL3[1] and LIL3[2] indicate monomeric and dimeric LIL3 (see Supplementary Fig. 3 for full-sized gels); PSII[1] and PSII[2]—monomeric and dimeric Photosystem II. Proteins separated in the second dimension were stained with Coomassie Blue (CBB). c SEC analysis of purified LIL3 and ELIP2. Chl absorbance and fluorescence of the separated proteins were monitored by diode-array and fluorescence detectors (Chl DAD and Chl FLD, respectively); both channels were normalized to their maxima. Fractions of 0.5 mL were collected, 20 μL of each separated by SDS-PAGE, blotted and probed by specified antibodies. Fractions collected within the grey rectangle were concentrated and used for pigment analysis (see later). d Pigments extracted from SEC fractions, indicated in grey, were quantified using HPLC; molar stoichiometries of the identified pigments are shown in parentheses. Values represent the means of three technical replicates; standard deviations were below 10%. Chl* indicates an unknown derivate of Chl. e Absorption spectra of LIL3 and ELIP2 proteins as recorded by a DAD during SEC and normalized to the Q_y peak.

Fig. 2. Characterization of the isolated Li-ELIP protein.

a A scheme of chimeric Li-ELIP protein; see also Supplementary Fig. 1 for the protein amino acid sequence. b Synechocystis membranes and the purified Li-ELIP were separated on CN-PAGE, 5 and 1.4 μg of Chl were loaded, respectively. The gel was scanned (Scan) and Chl fluorescence (Ch FL) was detected after excitation with blue light. Proteins were further separated by SDS-PAGE in the second dimension and the 2D gel was stained with Coomassie Blue (CBB). c SEC separation of Li-ELIP; Chl absorbance and fluorescence were monitored during chromatography by diode-array and fluorescence detectors (Chl DAD and Chl FLD). Fractions of 0.5 mL were collected, and those shown within the grey rectangle were concentrated and used for pigment analysis. d Extracted pigments were analysed by HPLC; molar stoichiometries of the identified pigments are shown in parentheses. Values represent means of three technical replicates; standard deviations were below 10%. e Absorption spectra of Li-ELIP and ELIP2 proteins as recorded by a DAD during SEC. f CD spectra in the visible region of LIL3 and Li-ELIP proteins. Data were scaled to the CD of the Q_y region at 660–690 nm.

Fig. 3. Ultrafast transient-absorption data measured after excitation of Chl in LIL3 and Li-ELIP.

Transient-absorption spectra of LIL3 (a) and Li-ELIP (b) immediately after excitation (dashed) and at 4 ps (solid). LIL3 and Li-ELIP proteins were excited at 670 nm and 675 nm, respectively. c The rise of the carotenoid signal after Chl excitation was measured at the maximum of the S₁–S_n band at 587 nm for LiL3 and 584 nm for Li-ELIP. d Decay of excited Chl monitored at 685 nm; the blue line shows Chl dynamics for unquenched ELIP2 for comparison. e Unified scheme of energy transfer pathways in LIL3 and Li-ELIP. The model assumes non-selective excitation of a pool of four Chl (red wavy arrow). The green double arrows represent the main Chl-to-carotenoid energy transfer quenching channel. Solid arrows denote relaxation processes associated either with the equilibration between Chl pools (blue) or with the decay of the carotenoid S₁ state (orange). The numbers correspond to the time constants (in ps) associated with each process in LIL3 (Li-ELIP). See Supplementary Fig. 8 for details.

Fig. 4. Analysis of the LIL3 and Li-ELIP proteins co-expressed with ZEP enzyme (ZEP⁺).

a The purified LIL3 (ZEP⁺) and Li-ELIP (ZEP⁺) protein variants were separated on 2D CN/SDS-PAGE; [1] and [2] indicate monomeric and dimeric forms, respectively. b SEC separation of Li-ELIP; Chl absorbance and fluorescence were monitored during chromatography by diode-array and fluorescence detectors (Chl DAD and Chl FLD). Fractions of 0.5 mL were collected and those shown within the grey rectangle were concentrated and used for pigment analysis. c Extracted pigments were analysed by HPLC; molar stoichiometries of the identified pigments are shown in parentheses. Values represent the means of three technical replicates; standard deviations were below 10%. A probable variant of pigment content per monomeric Li-ELIP protein is shown as an inset. d Absorption spectra of Li-ELIP (ZEP⁺), Li-ELIP, and ELIP2 proteins.

Fig. 5. Carotenoid specificity, dimerization, and mutagenesis of LHC-like proteins produced in Synechocystis.

a LIL3 was expressed in the Synechocystis xant⁻ background containing synechoxanthin and β-Car as the only carotenoids (see Supplementary Fig. 9a). Purified LIL3 was separated by 2D CN/SDS-PAGE and the gel stained by Coomassie Blue (CBB). b Solubilized membrane proteins isolated from the Synechocystis strain expressing LIL3 (LIL3 strain) and from the LIL3/xant⁻ were separated by 2D CN/SDS-PAGE. The SDS-gel was blotted, and the LIL3 protein was detected by a specific antibody. c Li-ELIP purified from the xant⁻ background was separated by 2D CN/SDS-PAGE and the gel stained by CBB. d A detail of lutein (lut1) binding site in pea LHCII protein (PDB code 2BHW). Chl molecules coordinated by E180 (Chl a1, forest green) and N183 residues (Chl a2, light green) close the lutein-binding cavity. Lutein is depicted as a yellow, space-filling model. e The purified LIL3-N177D protein was separated by 2D CN/SDS-PAGE, showing that the N177D mutation abolishes pigment binding. N177 residue in LIL3 corresponds to N183 Chl ligand in LHCII. f CD spectra in the UV region of LIL3 and the N177D LIL3 mutant normalized per residue (MRE mean residue ellipticity).

Fig. 6. A scheme of pigment binding to LIL3 protein.

LHC-like proteins, which contain only a single ‘Hlip helix’ (Hlips, OHPs, SEP/LIL3) have to dimerize to be able to associate with pigments. Although LIL3 is stable as a monomer (a) and can dimerize without pigments, the pigmentless dimer readily dissociates (b). A transient attachment of carotenoid molecules to LIL3 dimer is a precondition for the Chl-binding; in the absence of xanthophylls, Chl molecules cannot be ligated by the ExxNxR motif (c). On the other hand, the associated Chls enclose the carotenoid-binding cavity (see also Fig. 5d), and therefore the successive binding of carotenoids and Chls on each side of the dimer is essential for the establishment of a stable pigment-protein complex (d).