Nucleotide sequence of the hliA gene of Synechocystis sp. PCC6803 (hereinafter Synechocystis is used) was obtained from CyanoBase (http://genome.annotation.jp/cyanobase/Synechocystis/genes/ssl2542 ID ssl2542). This sequence was optimised for expression in bacteria and the synthetic gene was created. The gene was cloned into the pET28 vector in open reading frame with 6xHis at the C-terminus of resulting protein. E. coli BL(DE3) cells were transformed with obtained plasmid. HliA protein synthesis with His6 at the C-terminus (.....HGVIGWLNSL) 6xHis tag) was achieved by induction with IPTG. Recombinant protein was isolated from E. coli cells using metal-affinity chromatography on Ni-NTA Resin (Thermo-Fisher, USA) according to the manufacturer’s recommendation. Protein was stored at 4 °C in the presence of 1 mM sodium azide. Protein concentration was determined spectrophotometrically at 280 nm, using extinction coefficients from ProtParam programmes in ExPASy for calculation (https://web.expasy.org/protparam/).

After metal-affinity chromatography, fractions containing HliA protein (0.5–1 mg/ml) were dialysed against the buffer: 15mМ NaH₂PO₄ x 2H₂O, 0.07М KF, 15mМ urea, 0.1mM PMSF, рН 4.5 overnight at 4 °C and the protein concentration was measured spectrophotometrically using absorbance at 280 nm. Pigments (total chlorophyll a and carotenoids) were previously isolated from Synechocystis cells according to the method by Natali et al. (2014). The pigments were re-suspended in a buffer 15mМ NaH₂PO₄ x 2H₂O, 0.07М KF, 0.015М urea, 0.1mM PMSF, рН 4.5 and were sonicated on ice three times for 20 seconds with an interval of 10 seconds. Before the reconstitution, the HliA solution was subjected to short-term heating (95 °C) for 1 minute. Then the sample was transferred to ice and pigment extract was added in an equimolar ratio (1:1). After incubation on ice for 30 minutes, the sample was used for analysis.

Using SDS electrophoresis and subsequent Western blotting, the content (level) of recombinant HliA protein in E. coli cell lysate was evaluated (Akulinkina et al. 2015). Aliquots of HliA protein were separated in 12.5% SDS-polyacrylamide gel. The gel was stained by Coomassie R-250 or was used for immunodetection. The proteins were transferred from gel on to a nitrocellulose membrane (pore size of 45 mkm). Then the membrane with the transferred proteins was incubated for 1h at 4 °C in TBST buffer (50mM Tris-HCl, 200 mM NaCl, 0.1% Tween 20, pH 7.5), supplemented with 5% dry milk and then primary antibodies were added. We used polyclonal rabbit antibodies to HliA (1:4000) (Abcam, USA) or antibody in TBST buffer to 6X His-tag (1:5000) (ab213204, USA). The membrane was incubated with the antibodies overnight at 4 °C. The blots were rinsed and incubated with a secondary antibody – goat anti-rabbit IgG conjugated to horseradish peroxidase (1:2000) (Agrisera, Sweden). Immune complexes on a membrane were detected with the ECL fluorescent detection system (GE Healthcare England) and the signals were registered on X-ray film (Retina, Germany).

Fluorescence measurements were made on the Spectrofluorophotometer RF-5301 PC (Shimadzu, Japan) at 20 °C. Tryptophan residues make a decisive contribution to protein fluorescence. HliA protein contains two tryptophan residues. Fluorescence spectra of HliA protein (0.5 mg/ml) along with reconstituted complexes of protein with pigments in buffer (15mМ NaH₂PO₄ x 2H₂O, 0.07М KF, 15mМ urea, 0.1mM PMSF, рН 4.5) were excited at 295 nm (2 nm slit bandwidth) and were recorded in the range of 610–750 nm (5 nm slit bandwidth) at a speed of 200 nm/min.

The CD spectra of HliA protein (0.5 mg/ml) and reconstituted complexes of protein with pigments in buffer: 15mМ NaH₂PO₄ x 2H₂O, 0.07М KF, 15mМ urea, 0.1mM PMSF, рН 4.5, were performed on a Chirascan CD dichrograph (Applied Photophysics, UK) equipped with a thermal insert at 13 °C. The CD spectra were recorded in the range of 190–260 nm and 400–740 nm at a speed of 20 nm/min in a 0.02 cm quartz cuvette. Then the signal of buffer was subtracted from the spectra, prescribing its spectrum under identical conditions.

Purified recombinant HliA protein of Synechocystis was isolated by metal-affinity chromatography for the first time (Fig. 1A). The isolated protein had the expected molecular weight 10 kDa (Fig. 1A). The protein was present mainly in monomeric form. Sometimes a small number of HliA protein dimers have been detected. The isolation of HliA protein was confirmed by Western blotting using antibodies to HliA (Fig. 1B).

Obtaining purified HliA protein is of interest due to the possibility of studying its spectral properties, as well as studying its functions and interaction in vitro with pigments. This would serve as a direct proof of the binding of the pigment by protein. Most analyses, with the exception of structural analysis using X-ray crystallography or NMR, do not require the removal of the His-tag. Its presence does not have a significant impact on the result of the analysis. The method of circular dichroism spectroscopy was used to characterise the secondary structure of the HliA protein. The data of CD spectroscopy of HliA protein were processed using the DichroWeb programme (http://dichroweb.cryst.bbk.ac.uk/). It should be noted that there are relatively few spectra of membrane proteins in the CD database, which is due to their low solubility.

The CD spectrum in the far UV of the HliA protein preparation shows the presence of a secondary structure (Fig. 2) and corresponded to the alpha-helical structure of the protein. According to calculations, the alpha helix segments make up 61%. The crystallographic structure of the HliA protein and other Hli proteins has not yet been studied.

Pigments (chlorophyll a and carotenoids), isolated from Synechocystis, were used in HliA protein-binding experiments. It was planned to find out whether the HliA protein monomer has the ability to bind pigments.

Fluorescence spectra of HliA protein and HliA protein with pigments were studied (Fig. 3). As follows from the Figure, more intense fluorescence was shown for the sample of HliA protein with pigments then pure HliA protein. This may be due to the binding of the pigment to the formation of the complex.

Then the CD spectra of pigments (chlorophyll and carotenoids), HliA protein and their mixture were studied. A change in the CD spectra in the far red (640–740 nm) region of the HliA protein, incubated with pigments in comparison with the control pigments sample, was detected. This result indicates the interaction of chlorophyll with HliA protein. Small signal changes in the CD spectra in the 480–507 nm region were also detected, indicating the interaction of HliA protein with carotenoids (https://conf.icgbio.ru/plantgen2021/en/2021/06/06/detection-of-the-binding-the-stress-hlia-protein-synechocystis-sp-with-pigments/). Apparently, no more than 1–2 molecules of chlorophyll and carotenoids bind to the protein. Analysis of CD spectra of the HliA protein with pigments showed that the sample contained the monomeric chlorophyll. Thus, it has been shown experimentally that HliA protein molecules have the ability to bind pigments.