BioRisk | of | 39- | 46 (2022) Apeer-reviewed open-access journal

doi: 10.3897/biorisk.17.77306 RESEARCH ARTICLE & B te) Re IS k

https://biorisk.pensoft.net

In vitro reconstitution of complexes
of stress HIiA protein with pigments

Lubov Sharapova', Nadezhda Yurina'

| Buch Institute of Biochemistry, Federal Research Center of Biotechnology of Russian Academy of Sciences,
Moskow, Russia

Corresponding author: Nadezhda Yurina (nadezhdayurina@hotmail.com)

Academic editor: S. Chankova | Received 29 October 2021 | Accepted 7 December 2021 | Published 21 April 2022

Citation: Sharapova L, Yurina N (2022) In vitro reconstitution of complexes of stress HIiA protein with pigments.
In: Chankova S, Peneva V, Metcheva R, Beltcheva M, Vassilev K, Radeva G, Danova K (Eds) Current trends of ecology.
BioRisk 17: 19-146. https://doi.org/10.3897/biorisk.17.77306

Abstract

Proteins similar to Hli (high light inducible) proteins of cyanobacteria are present in all photosynthetic
eukaryotes and are necessary for survival in various stressful conditions, although their exact function is
not fully understood. In current study, the recombinant stress-induced protein HliA of cyanobacterium
Synechocystis was isolated and characterised for the first time. The synthetic gene of HIiA protein was
created and cloned into plasmid for expression of recombinant protein with Hisx6-tag at the C-terminus
in bacteria. Recombinant HIiA protein of Synechocystis was isolated by metal-afhnity chromatography. The
HIiA protein was reconstituted with chlorophyll a and carotenoids. Using circular dichroism spectroscopy,
it was shown that chlorophyll a and carotenoids interact in vitro with the HIiA protein. The binding of
pigments to the HIiA protein favours the protective function of this protein. Apparently, Hli proteins are
involved in the coordinated delivery of pigments for the biogenesis of photosynthetic complexes, thereby
reducing the risk of accumulation of phototoxic free chlorophyll molecules. Current results are important

for understanding the processes of photoprotection in either cyanobacteria or algae and higher plants.

Keywords
Carotenoid, chlorophyll, cyanobacteria, high light inducible proteins, high light stress, Synechocystis

Copyright Lubov Sharapova & Nadezhda Yurina. This is an open access article distributed under the terms of the Creative Commons Attribution License
(CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

140 Lubov Sharapova & Nadezhda Yurina / BioRisk 17: 139-146 (2022)

Introduction

Cyanobacteria are ubiquitous and inhabit almost all ecosystems, thanks to their effec-
tive adaptation to various conditions, including extreme ones. Therefore, these pho-
totrophs are a promising model system for studying mechanisms of resistance and
adaptation to various stresses, including one of the most common — light stress.

Under light stress, the photosynthetic apparatus is damaged due to the accumula-
tion of both toxic free chlorophyll molecules and the formation of reactive oxygen
species. For the normal functioning of photosynthetic organisms under conditions of
light stress, numerous protective mechanisms have emerged in the course of evolution.
Protective mechanisms involving light-induced stress proteins are of great importance.
These include cyanobacterial Hlips (high light-inducing proteins) with a single trans-
membrane helix (Chidgey et al. 2014; Hey and Grimm 2020).

Hli proteins are necessary for cell survival under light stress and other stressful
conditions, as well as for maintaining normal cell activity. It is assumed that Hi proteins
take part in important processes, such as: regulation of chlorophyll biosynthesis;
transport and binding of free chlorophyll molecules; quenching of singlet oxygen;
assembly and repair of photosystem 2; non-photochemical dissipation of absorbed light
energy (Komenda and Sobotka 2016). However, the full picture of the functioning of
Hli proteins has not been fully studied.

Four Hli proteins (HIiA, B, C and D) were found in the unicellular cyanobacterium
Synechocystis sp. PCC 6803 (Staleva et al. 2015). These proteins have been isolated
as part of small complexes with other proteins and only recently, the first individual
HliC protein from this family has been purified (Shukla et al. 2018). There is no such
information about other representatives of Hli proteins. Of particular interest is the
protein of this family — HIiA, since it is important for the survival of Synechosystis cells
under light stress (He et al. 2001). To protect cells from excessive light, binding of
toxic free chlorophyll molecules, which cause photodestruction and oxidative stress, is
necessary. The stress protein HIiA can serve as such a key agent binding these molecules.
Therefore, to understand this process, it is necessary to obtain a recombinant HliA
protein and its subsequent complex characterisation and study of its interaction with
chlorophyll iz vitro. Isolation and characterisation of pure HIiA protein has not yet
been carried out. Until recently, it was not clear whether the pigments (chlorophyll a
and beta-carotene) bind to the HIiA protein.

The aim of the study was to isolate pure recombinant HIiA protein and reconstruct
the HIiA protein complex with pigments (chlorophyll a and carotenoids).

Materials and methods

HIiA gene expression and protein purification

Nucleotide sequence of the ALA gene of Synechocystis sp. PCC6803 (hereinafter

Synechocystis is used) was obtained from CyanoBase (http://genome.annotation.jp/

In vitro reconstitution of complexes of stress HIiA protein with pigments 141

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. HIiA 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-afhnity 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 coefhicients from ProtParam programmes in ExPASy for
calculation (https://web.expasy.org/protparam/).

The reconstitution of complexes of HIliA-6xHis protein and pigments

After metal-afhinity chromatography, fractions containing HIiA protein (0.5—1 mg/
ml) were dialysed against the buffer: 15mM NaH,PO, x 2H,O, 0.07M KF, 15mM
urea, 0.1mM PMSF, pH 4.5 overnight at 4 °C and the protein concentration was
measured spectrophotometrically using absorbance at 280 nm. Pigments (total chlo-
rophyll 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
15mM NaH,PO, x 2H,O, 0.07M KF, 0.015M urea, 0.1mM PMSF, pH 4.5 and
were sonicated on ice three times for 20 seconds with an interval of 10 seconds.
Before the reconstitution, the HIiA 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.

Western blotting

Using SDS electrophoresis and subsequent Western blotting, the content (level) of
recombinant HIiA protein in E.coli cell lysate was evaluated (Akulinkina et al. 2015).
Aliquots of HIiA 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 HIiA (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).

142 Lubov Sharapova & Nadezhda Yurina / BioRisk 17: 139-146 (2022)

Fluorescence spectroscopy

Fluorescence measurements were made on the Spectrofluorophotometer RF-5301 PC
(Shimadzu, Japan) at 20 °C. Tryptophan residues make a decisive contribution to pro-
tein fluorescence. HIiA protein contains two tryptophan residues. Fluorescence spectra
of HIiA protein (0.5 mg/ml) along with reconstituted complexes of protein with pig-
ments in buffer (15mM NaH,PO, x 2H,O, 0.07M KE, 15mM urea, 0.1mM PMSF,
pH 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.

Circular dichroism spectroscopy

The CD spectra of HIiA protein (0.5 mg/ml) and reconstituted complexes of
protein with pigments in buffer: 15mM NaH,,PO, x 2H,O, 0.07M KF, 15mM urea,
0.lmM PMSE, pH 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.

Results

Isolation and purification of HliA-6xHis protein

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

Obtaining purified HIiA 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 signifi-
cant impact on the result of the analysis. The method of circular dichroism spectros-
copy was used to characterise the secondary structure of the HIiA protein. The data of
CD spectroscopy of HIiA 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 HIiA 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 crystal-
lographic structure of the HIiA protein and other Hi proteins has not yet been studied.

In vitro reconstitution of complexes of stress HIiA protein with pigments 143

B
1 2 , «££ 5 8 F% M en,
250
70
| 25
15
— 10
Pa

Figure |. SDS-PAGE (A) and immunoblot analysis of the HIiA eluate A fraction collected during His-
tag affinity chromatography (separation) of HIiA eluate were further separated by SDS-electrophoresis
and were stained with Coomassie brilliant blue. The numbers at the top of the gel indicate elution fraction
numbers from the column. The arrows show the location of the HIiA protein in the gel B the gel was
blotted and the HIiA protein immunodetected.

Absorption

220 240
Wavelength, nm Wavelength, nm

Figure 2. Circular dichroism A and absorption B spectra of the HIiA protein.

Reconstitution of complexes of stress HIiA protein with pigments

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

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

144 Lubov Sharapova & Nadezhda Yurina / BioRisk 17: 139-146 (2022)

— HIiA
HliA+pigments

c
2
D
2
£
uw

700
Wavelength, nm

Figure 3. Fluorescence emission of reconstituted pigment-HIiA protein (---) and HIiA protein (—) samples.

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

Discussion

In previously published papers, the HIiA protein was studied in complex with other Hli
proteins. In this work, the pure recombinant protein HIliA Synechocystis was isolated for
the first time. The pigment-binding capacity of the HliA protein was studied and the
reconstruction of the HIiA protein with pigments was carried out. CD spectroscopy
has shown that chlorophyll a and carotenoids interact im vitro with the HIiA protein.
The data obtained by us are consistent with the fact that Hli proteins contain
conserved amino acid residues that are important for the binding of chlorophyll in

In vitro reconstitution of complexes of stress HIliA protein with pigments 145

all proteins of light-harvesting complexes (Shukla et al. 2018). The HIiA protein also
contains a chlorophyll binding domain.

It is known that free chlorophyll molecules in a cell are dangerous for cells due to
the threat of the formation of damaging reactive oxygen species. In this regard, the
binding of pigments to the HIiA protein revealed by us indicates the protective role
of this protein. Apparently, Hli proteins can participate in the coordinated delivery of
pigments in the biogenesis of photosynthetic complexes, reducing the risk of accumu-
lation of phototoxic unrelated chlorophyll molecules (Shukla et al. 2018).

We found a smaller number of bound chlorophyll molecules (1-2) per protein
than was hypothetically assumed (4—5 molecules). Perhaps this is due to the fact that a
complex with other protein molecules is needed to bind more chlorophyll molecules.
Apparently, the association of HIiA protein with lipid components of the membrane
is necessary for the binding of a large quantity of pigment molecules. It is possible
that the binding of pigments depends on the formation of HIiA/HIiB heterodimers
or HIiA protein homodimers. It is assumed that Hli proteins are important for the re-
utilisation of free chlorophyll molecules (Shukla et al. 2018).

The study of the constitution of the recombinant HIiA protein with chlorophyll
a and carotenoids in vitro allows us to expand knowledge about the photoprotective
functions of light-induced Hli proteins. The results are important for understanding
photoprotection processes in both cyanobacteria and algae and higher plants.
Knowledge about the mechanism of functioning of stress proteins can also be used in
the creation of artificial photosynthesis systems.

Conclusion

Pure recombinant protein HIiA Synechocystis was isolated by Ni-afhinity chromatography.
The binding of pigments to HliA was shown, which confirms the protective function
of this protein. Apparently, HIiA proteins are involved in the coordinated delivery of
pigments in the biogenesis of photosynthetic complexes, reducing the risk ofaccumulation
of phototoxic free chlorophyll molecules. ‘The results are important for understanding
photoprotection processes in both cyanobacteria and algae and higher plants.

Acknowledgements

This study was partially supported by the Russian Foundation for Basic Research (Pro-
ject N 19-04- 00798 “Complex characterization of the cyanobacterial high-light-in-
ducible HIiA protein to understand the mechanism of photoprotection”). We would
like to extend our thanks to Dr. Yu.V. Bolychevtseva for assistance rendered in fluo-
rescence measurements. The equipment and software for mass spectrometry at the
Industrial Biotechnologies Center for Collective Use of the Federal Research Center of
Biotechnology of the Russian Academy of Sciences were used.

146 Lubov Sharapova & Nadezhda Yurina / BioRisk 17: 139-146 (2022)

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