Stress Protein HliA Shown to Capture Toxic Chlorophyll in Lab Reconstitution

When sunlight becomes too intense, the machinery that photosynthetic organisms rely on for life can turn against them. Excess light energy drives the formation of reactive oxygen species, and free chlorophyll molecules that have not yet been safely installed into photosynthetic complexes can wreak havoc inside the cell. A family of tiny proteins known as high light-inducible proteins, or Hlips, has long been suspected of standing guard against this damage. Now, researchers have taken a crucial step toward understanding exactly how one of these guardians works, isolating a single Hlip protein in pure form for the first time and demonstrating directly in the test tube that it grabs hold of chlorophyll and carotenoids.

The focus of the new study, published in the journal BioRisk, is HliA, one of four high light-inducible proteins found in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Hlips are remarkable in their reach: proteins similar to them are present in all photosynthetic eukaryotes, from algae to higher plants, and they are required for survival under a range of stressful conditions. Each Hlip carries just a single transmembrane helix, making it one of the smallest members of the vast family of chlorophyll-binding proteins. Yet despite decades of research, their precise molecular functions have remained only partially understood, with proposed roles ranging from regulating chlorophyll biosynthesis to quenching singlet oxygen and assisting the assembly and repair of photosystem 2.

Why focus on HliA in particular? Among the four Synechocystis Hlips, HliA stands out as critical for cell survival under light stress. Previous work had only ever isolated these proteins as parts of small complexes with other Hli family members; just one individual representative, HliC, had been purified before. Without a pure protein in hand, researchers could not directly test a central question: does HliA actually bind the toxic free chlorophyll molecules that accumulate when light overwhelms the photosynthetic apparatus? Answering that question required producing the protein from scratch in the laboratory.

The team began with the hliA gene sequence from Synechocystis, retrieved from the CyanoBase genome database. Because bacteria do not always express cyanobacterial genes efficiently, the researchers chemically synthesized a version of the gene whose nucleotide sequence was optimized for expression in Escherichia coli. They cloned this synthetic gene into a pET plasmid vector, arranging it so that the resulting protein carried a tag of six histidine residues at its C-terminus. This His-tag is a standard molecular handle: it binds tightly to nickel ions, allowing the recombinant protein to be fished out of bacterial cell lysates by metal-affinity chromatography on nickel-NTA resin. After induction of protein synthesis with IPTG and purification, the team obtained a protein of the expected molecular weight of about 10 kilodaltons, present mostly in monomeric form with occasional dimers. Western blotting, using antibodies raised against HliA and against the His-tag, confirmed the identity of the isolated protein.

With pure HliA available, the researchers turned to circular dichroism spectroscopy to characterize its secondary structure. This technique measures how proteins circularly polarize absorbed light, and the resulting spectra in the far ultraviolet reveal the proportions of alpha helices and other structural elements. The spectrum of HliA corresponded clearly to an alpha-helical fold, with calculations indicating that roughly 61 percent of the protein is arranged in alpha-helical segments. That figure makes sense for a protein built around a single transmembrane helix, and it aligns with Hlips belonging to the light-harvesting complex protein superfamily, whose members share conserved chlorophyll-binding residues. No crystallographic or NMR structure of HliA or its relatives exists yet, so spectroscopic characterization provides a valuable first glimpse of the protein’s architecture.

The centerpiece of the study was the reconstitution experiment. The researchers isolated chlorophyll a and carotenoids from Synechocystis cells and prepared them in an acidic buffer, sonicating the pigment extract on ice to disperse it. The HliA protein, briefly heated to 95 degrees Celsius and then chilled, was mixed with the pigments in a one-to-one molar ratio and incubated on ice. Fluorescence spectroscopy provided the first hint of success: samples containing both protein and pigments showed more intense fluorescence than the protein alone, consistent with complex formation between HliA and its pigment cargo. Because the protein contains two tryptophan residues whose fluorescence was excited at 295 nanometers, the technique could sensitively report on changes in the protein’s environment upon pigment binding.

Circular dichroism spectroscopy then delivered the most direct evidence. In the far-red region of the spectrum, between 640 and 740 nanometers, the reconstituted samples showed altered CD signals compared with pigments alone, a signature of chlorophyll interacting with the protein. Smaller signal changes appeared in the 480 to 507 nanometer region, indicating interaction with carotenoids as well. Importantly, analysis of the spectra showed that the chlorophyll in these complexes was monomeric rather than aggregated, suggesting that HliA keeps individual chlorophyll molecules in a controlled state. The stoichiometry came as something of a surprise: only about one or two chlorophyll molecules appeared to bind per protein, fewer than the four to five that had been hypothetically proposed. The researchers suggest that capturing more pigment may require HliA to associate with other partners, such as lipid components of the membrane, or to form homodimers or HliA-HliB heterodimers.

The findings carry weight beyond the test tube. Free chlorophyll molecules are dangerous intracellular agents because, once excited by light, they can transfer energy to oxygen and generate reactive oxygen species that destroy membranes, proteins, and DNA. By demonstrating that HliA binds these molecules directly, the study supports the idea that Hlips act as photoprotective scavengers and as chaperones that deliver pigments in a coordinated fashion during the biogenesis of photosynthetic complexes, preventing phototoxic free chlorophyll from piling up. This is consistent with earlier observations that Hlips share conserved amino acid residues important for chlorophyll binding across the entire light-harvesting complex protein family, and that HliA contains a recognizable chlorophyll-binding domain.

The implications ripple outward from cyanobacteria to agriculture and synthetic biology. Since Hlip-like proteins are conserved across algae and higher plants, understanding their pigment-binding mechanism illuminates photoprotection strategies used by virtually all oxygen-producing photosynthetic organisms, knowledge that could inform the engineering of crops with improved stress tolerance. The work also offers lessons for designers of artificial photosynthesis systems, where safely handling reactive chlorophyll-like pigments is a central challenge. For now, the purified HliA protein and its reconstituted pigment complexes provide a new experimental platform, one that researchers can use to dissect, molecule by molecule, how the smallest guardians of the photosynthetic world keep toxic light in check.

Subject of Research: In vitro reconstitution and pigment binding of the cyanobacterial stress protein HliA

Article Title: In vitro reconstitution of complexes of stress HliA protein with pigments

Article References: Sharapova, L., & Yurina, N. (2022). In vitro reconstitution of complexes of stress HliA protein with pigments. BioRisk, 17, 139-146. https://doi.org/10.3897/biorisk.17.77306

Image Credits: AI Generated

DOI: 10.3897/biorisk.17.77306

Keywords: cyanobacteria, HliA, high light-inducible proteins, chlorophyll a, carotenoids, photoprotection, circular dichroism spectroscopy, Synechocystis sp. PCC 6803, recombinant protein, reactive oxygen species, photosynthesis, light stress