Biology subjects

Maksimov, E. G.

Publications and source records attributed to Maksimov, E. G..

At least 19 recordsLinked to original sources

bioRxiv2025

Alteration in the intracellular Na+/K+ ratio regulates gene expression through the shift of G-quadruplex dynamics in living cells

Alteration in the intracellular Na+i/K+i ratio can play an important regulatory role in many mammalian cells. Here, we demonstrated that the expression of some early response genes does depend on the intracellular Na+i/K+i ratio and that the inhibition of DNA-binding activity of transcription factors c-Fos and c-Jun attenuates the upregulation of Zfp36 and Egr1 genes in HeLa cells. Both replacement of intracellular Na+ and K+ by Li+ and the dissipation of Na+i/K+i gradient demonstrated a similar effect on the transcription of Fos, Jun, Zfp36, and Ptgs2. Oligonucleotide sequences from the Fos, Kit, and My[c] promoters with an ability to fold into G-quadruplexes (G4s) change their conformations differently depending on the ratio of Na+, K+, and Li+ ions in vitro. Fluorescence lifetime imaging microscopy studies using DNA G4-specific dye DAOTA-M2 revealed that changes in compositions and ratios of monovalent metal cations in HeLa cells were accompanied by alterations in intranuclear G4s dynamics. The data demonstrate that these structures may be considered as intracellular sensors of monovalent metal cations regulating gene expression through the shift of G4s dynamics.

biochemistry↗

bioRxiv2025

Architecture of the bundle-shaped phycobilisome from Gloeobacter violaceus

Cyanobacteria use soluble antenna megacomplexes, phycobilisomes (PBS), to maximize light-harvesting efficiency and small photoswitchable orange carotenoid proteins (OCPs) to down-regulate PBS in high light. Among known PBS morphologies, the one from the basal cyanobacterial genus Gloeobacter still lacks detailed structural characterization. Here, we present the cryo-EM structure of the G. violaceus PBS, a >10-MDa complex with a unique architecture consisting in 516 polypeptide chains totaling nearly 90,000 residues and harboring over 860 bilin chromophores. This unique PBS features diverging, conformationally mobile bundles of rods composed of stacked phycoerythrin and phycocyanin hexamers, stemming from a pentacylindrical allophycocyanin core belted by auxiliary phycocyanin hexamers. We show how two Gloeobacter-specific multidomain linker proteins, Glr1262 and Glr2806, maintain this bundle-shaped architecture and reveal its differential regulation via non-photochemical quenching by two OCP types of G. violaceus that recognize separate binding sites within the allophycocyanin core, including lateral cylinders absent in tricylindrical cores. Our findings provide the high-resolution structural insight into Gloeobacter PBS and its regulation, revealing divergent adaptations in early-branching cyanobacteria. The structure advances understanding of PBS diversity and evolution, offering a framework for bioengineering light-harvesting systems in synthetic biology and biotechnological applications.

biochemistry↗

bioRxiv2025

Discovery of excited state proton transfer in flavin-based fluorescent protein with large Stokes shift

Flavin-binding proteins (flavoproteins) are widespread in nature, revealing versatile oxidation-reduction reactions and photochemistry. Flavoproteins derived from LOV domains are used for engineering of ligh-tresponsive tools in optogenetics, as well as fluorescent markers and photogenerators of reactive oxygen species. Despite extensiev efforts, all currently used LOV-derived proteins have similar absorption spectra with maxima around 275, 35-0375, and 450-485 nm. Here, we describe the discovery of a large Stokes shift flavi-nbased fluorescent protein, LSSFbFP, which can be obtainedin vivo and in vitro, with absorption maxima at 340-350 and 395-405 nm. Fluorescence emission of LSSFbFP mirrors that of classical FbFPs with the maximum at ~500 nm. We sho that the protein binds lumichrome as the chromophore and use low temperature and time-resolved spectroscopy, X-ray crystallography and modeling to prove that the apparent Stokes shift of LSSFbFP occurs due to excited state proton phenomena observed in flavoproteni s and pave the way for engineering of new flavin-based molecular instruments.

biochemistry↗

bioRxiv2023

Structural framework for the understanding spectroscopic and functional signatures of the cyanobacterial Orange Carotenoid Protein families

The Orange Carotenoid Protein (OCP) is a unique photoreceptor crucial for cyanobacterial photoprotection. Best studied Synechocystis sp. PCC 6803 OCP belongs to the large OCP1 family. Downregulated by the Fluorescence Recovery Protein (FRP) in low-light, high-light-activated OCP1 binds to the phycobilisomes and performs non-photochemical quenching. Recently discovered families OCP2 and OCP3 remain structurally and functionally underexplored, and no systematic comparative studies have ever been conducted. Here we present two first crystal structures of OCP2 from morphoecophysiologically different cyanobacteria and provide their comprehensive structural, spectroscopic and functional comparison with OCP1, the recently described OCP3 and all-OCP ancestor. Structures enable correlation of spectroscopic signatures with the effective number of hydrogen and discovered here chalcogen bonds anchoring the ketocarotenoid in OCP and rationalize the observed differences in OCP/FRP and OCP/phycobilisome functional interactions. These data are expected to foster OCP research and applications in optogenetics, targeted carotenoid delivery and cyanobacterial biomass engineering.

biochemistry↗

bioRxiv2023

Two distinct mechanisms of flavoprotein spectral tuning revealed by low-temperature and time-dependent spectroscopy

Flavins such as flavin mononucleotide or flavin adenine dinucleotide are bound by diverse proteins, yet have very similar spectra when in the oxidized state. Recently, we developed new variants of flavin-binding protein CagFbFP exhibiting notable blue (Q148V) or red (I52V A85Q) shifts of fluorescence emission maxima. Here, we use time-resolved and low temperature spectroscopy to show that whereas the chromophore environment is static in Q148V, an additional protein-flavin hydrogen bond is formed upon photoexcitation in the I52V A85Q variant. Consequently, in Q148V, excitation, emission and phosphorescence spectra are shifted, whereas in I52V A85Q, excitation and low-temperature phosphorescence spectra are relatively unchanged, while emission spectrum is altered. We also determine X-ray structures of the two variants to reveal the flavin environment and complement the spectroscopy data. Our findings illustrate two distinct color tuning mechanisms of flavin-binding proteins and pave the way for engineering of new variants with improved optical properties. TOC GRAPHICS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/549366v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1403d8eorg.highwire.dtl.DTLVardef@894bd1org.highwire.dtl.DTLVardef@1d089b1org.highwire.dtl.DTLVardef@1ffd3c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Structural basis for the ligand promiscuity of the neofunctionalized, carotenoid-binding fasciclin domain protein AstaP

Fasciclins (FAS1) are ancient adhesion protein domains found across different phyla from bacteria to humans, with no common small ligand binding function reported. A unique FAS1-containing astaxanthin-binding protein (AstaP) from green algae can efficiently bind an unusually broad repertoire of carotenoids (astaxanthin, zeaxanthin, canthaxanthin, {beta}-carotene), but the underlying mechanism is largely unknown. Here we dissect the structural basis for the ligand binding promiscuity of AstaP-orange1 (AstaPo1) by determining its solution NMR structure in complex with its natural ligand, astaxanthin (AXT), and validate this structure by SAXS, calorimetry, optical spectroscopy and mutagenesis data. While the unstructured tails of AstaPo1 are not essential for carotenoid binding, they enhance protein solubility. The a1-a2 helices of the AstaPo1 FAS1 domain embrace the carotenoid polyene like a jaw, organizing a conserved hydrophobic tunnel, too short to prevent the AXT {beta}-ionone rings from protruding on both sides of the tunnel, thereby not imposing specificity restrictions. The only specific protein-AXT interactions involve H-bonds between the oxygenated groups on AXT and a peripheral Gln56 residue. Remarkably, mapping of this and other AXT-contacting AstaPo1 residues revealed their different conservation in AstaP orthologs with the tentative carotenoid-binding function and in FAS1 proteins in general, supporting neofunctionalization of AstaPs within green algae. Correspondingly, a cyanobacterial homolog with a similar domain structure cannot bind carotenoids due to subtle differences in residues decorating the tunnel. These structure-activity relationships inform the sequence-based prediction of the carotenoid-binding FAS1 members. SIGNIFICANCEA water-soluble astaxanthin-binding protein (AstaP) is a photoprotective protein in green algae helping them to tolerate stress conditions. While belonging to a ubiquitous protein family sharing an ancient structural domain, fasciclin, involved in cell adhesion, AstaP possesses an outstanding ability to bind carotenoid pigments of a different type, which are potent antioxidants. To understand the molecular basis for such carotenoid-binding promiscuity of AstaP, here we determined its spatial structure - the first structure of a carotenoid-protein complex solved by nuclear magnetic resonance spectroscopy. Together with biochemical and sequence conservation analyses, our data illustrate a remarkable case of neofunctionalization of the ancient protein domain and pave the way for its bioengineering and practical use as antioxidant transporter for biomedical applications.

biochemistry↗

bioRxiv2022

Nanocontainer derived from silkworm carotenoprotein for carotenoid extraction and presentation in biotechnology and biomedical applications

Found in many organisms, soluble carotenoproteins are considered as antioxidant nanocarriers for biomedical applications, although the structural basis for their carotenoid transfer function, a prerequisite for rational bioengineering, is largely unknown. We report crystal structures of the Carotenoid-Binding Protein from Bombyx mori (BmCBP) in apo- and zeaxanthin (ZEA)-bound forms. We use spectroscopy and calorimetry to characterize how ZEA and BmCBP mutually affect each other in the complex, identify key carotenoid-binding residues, confirm their roles by crystallography and carotenoid-binding capacity of BmCBP mutants and reconstitute BmCBP complexes with biomedically-relevant xanthophylls lutein, zeaxanthin, canthaxanthin and astaxanthin. By cost-effectively and scalably solubilizing xanthophylls from various crude herbal extracts, His-tagged BmCBP remains monomeric and forms a dynamic nanocontainer delivering carotenoids to liposomes and to other carotenoid-binding proteins, which in particular makes the Orange Carotenoid Protein, a promising optogenetic tool, photoactive. Furthermore, BmCBP(ZEA) administration stimulates fibroblast growth, which paves the way for its biomedical applications.

biochemistry↗

bioRxiv2022

Crystal structure and functional peculiarities of a primordial Orange Carotenoid Protein (OCPX)

The two-domain photoactive Orange Carotenoid Protein (OCP) confers photoprotection in cyanobacteria and presumably stems from domain fusion. Yet, the primitive thylakoid-less cyanobacteria Gloeobacter encodes a complete OCP. Its photosynthesis regulation lacks the so-called Fluorescence Recovery Protein (FRP), which in Synechocystis inhibits OCP-mediated phycobilisome fluorescence quenching, and Gloeobacter OCP belongs to the recently defined, heterogeneous clade OCPX (GlOCPX), the least characterized compared to OCP2 and especially OCP1 clades. Here we describe the first crystal structure of OCPX and provide its detailed structural and functional comparison with OCP1 from Synechocystis. Monomeric GlOCPX quenches Synechocystis phycobilisomes but displays drastically accelerated, less temperature-dependent recovery after photoactivation, evades regulation by FRP from other species and reveals numerous structural features reflecting its functional peculiarities. Our detailed description of a primordial OCPX sheds light on the evolution of the OCP-dependent photoprotection mechanism, rationalizing subdivision of the OCPX clade into subclades.

biochemistry↗

bioRxiv2021

Microalgal protein AstaP is a potent carotenoid solubilizer and delivery module with a broad carotenoid binding repertoire

Carotenoids are lipophilic substances with many biological functions, from coloration to photoprotection. Being potent antioxidants, carotenoids have multiple biomedical applications, including the treatment of neurodegenerative disorders and retina degeneration. Nevertheless, the delivery of carotenoids is substantially limited by their poor solubility in the aqueous phase. Natural water-soluble carotenoproteins can facilitate this task, necessitating studies on their ability to uptake and deliver carotenoids. One such promising carotenoprotein, AstaP (Astaxanthin-binding protein), was recently identified in eukaryotic microalgae, but its structure and functional properties remained largely uncharacterized. By using a correctly folded recombinant protein, here we show that AstaP is an efficient carotenoid solubilizer that can stably bind not only astaxanthin but also zeaxanthin, canthaxanthin, and, to a lesser extent, {beta}-carotene, i.e. carotenoids especially valuable to human health. AstaP accepts carotenoids provided as acetone solutions or embedded in membranes, forming carotenoid-protein complexes with an apparent stoichiometry of 1:1. We successfully produced AstaP holoproteins in specific carotenoid-producing strains of Escherichia coli, proving it is amenable to cost-efficient biotechnology processes. Regardless of the carotenoid type, AstaP remains monomeric in both apo- and holoforms, while its rather minimalistic mass ([~]20 kDa) makes it an especially attractive antioxidant delivery module. In vitro, AstaP transfers different carotenoids to the liposomes and to unrelated proteins from cyanobacteria, which can modulate their photoactivity and/or oligomerization. These findings expand the toolkit of the characterized carotenoid-binding proteins and outline the perspective of the use of AstaP as a unique monomeric antioxidant nanocarrier with an extensive carotenoid-binding repertoire.

biochemistry↗

bioRxiv2021

Antigen-specific stimulation and expansion of CAR-T cells using membrane vesicles as target cell surrogates

Development of CAR-T therapy led to immediate success in the treatment of B cell leukemia and lymphoma. It also raised an opportunity to design new protocols to target solid tumors. Manufacturing of therapy-competent functional CAR-T cells needs robust protocols for ex vivo/in vitro expansion of modified T-cells. This step is challenging, especially if non-viral low efficiency delivery protocols are used to generate CAR-T cells. Modern protocols for CAR-T cell expansion are based on incubation with high doses of recombinant cytokines to support proliferation, non-specific stimulation with surface-bound antibodies to induce TCR cross-linking, or co-cultivation with antigen-expressing feeder cell lines. These approaches are imperfect since non-specific stimulation results in rapid outgrowth of CAR-negative T cells, and removal of feeder cells from mixed cultures necessitates additional purification steps. In an effort to develop a specific and improved protocol for CAR-T cell expansion, we took advantage of cell-derived membrane vesicles, and the simple structural demands of the CAR-antigen interaction. Our approach was to make antigenic microcytospheres from common cell lines stably expressing surface-bound CAR antigens (antigenic vesicles, AVs), and then use them for stimulation and expansion of CAR-T cells. We developed a rapid, simple, efficient, and inexpensive protocol to generate, stabilize and purify AVs. As proof-of-concept we tested the efficacy of our AV constructs on several CAR-antigen pairs. The data presented in this article clearly demonstrate that our protocol produced AVs with the capacity to induce stronger stimulation, proliferation and functional activity of CAR-T cells than is possible with existing protocols. We predict that this new methodology will significantly improve the ability to obtain improved populations of functional CAR-T cells for therapy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/435976v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@abbe23org.highwire.dtl.DTLVardef@15007dforg.highwire.dtl.DTLVardef@134e506org.highwire.dtl.DTLVardef@1a7f1a2_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2020

Molecular basis for the recognition of steroidogenic acute regulatory protein by the 14-3-3 protein family

Steroidogenesis in adrenals and gonads starts from cholesterol transport to mitochondria by the steroidogenic acute regulatory protein STARD1, containing a mitochondrial import sequence followed by a cholesterol-binding START domain. Although mutations in this protein have been linked to lipoid congenital adrenal hyperplasia, the mechanism of steroidogenesis regulation by the STARD1 remains debatable, hypothetically involving a molten-globule structural transition and interaction with 14-3-3 proteins. We show that, while the isolated START domain does not interact with 14-3-3, interaction is enabled by STARD1 phosphorylation at Ser57, close to the mitochondrial peptide cleavage site. Biochemical analysis of the STARD1 affinity towards 14-3-3 and crystal structures of 14-3-3 complexes with Ser57 and Ser195 phosphopeptides, suggest distinct roles of site-specific phosphorylations in recruiting 14-3-3, to modulate STARD1 activity, processing and import to mitochondria. Phosphorylation at Ser195 creates a unique conditional site, that could only bind to 14-3-3 upon partial unfolding of the START domain.

biochemistry↗

bioRxiv2019

Soluble cyanobacterial carotenoprotein as a robust antioxidant nanocarrier and delivery module

To counteract oxidative stress, antioxidants including carotenoids are highly promising, yet their exploitation is drastically limited by the poor bioavailability and fast photodestruction, whereas current delivery systems are far from being efficient. Here we demonstrate that the recently discovered nanometer-sized water-soluble carotenoprotein from Anabaena (termed CTDH) transiently interacts with liposomes to efficiently extract carotenoids via carotenoid-mediated homodimerization, yielding violet-purple protein samples amenable to lyophilization and long-term storage. We characterize spectroscopic properties of the pigment-protein complexes and thermodynamics of liposome-protein carotenoid transfer and demonstrate the highly efficient delivery of echinenone form CTDH into liposomes. Most importantly, we show carotenoid delivery to membranes of mammalian cells, which provides protection from reactive oxygen species. The described carotenoprotein may be considered as part of modular systems for the targeted antioxidant delivery.\n\nSignificance statementCarotenoids are excellent natural antioxidants but their delivery to vulnerable cells is challenging due to their hydrophobic nature and susceptibility to degradation. Thus, systems securing antioxidant stability and facilitating targeted delivery are of great interest for the design of medical agents. In this work, we have demonstrated that soluble cyanobacterial carotenoprotein can deliver echinenone into membranes of liposomes and mammalian cells with almost 70 % efficiency, which alleviates the induced oxidative stress. Our findings warrant the robustness of the protein-based carotenoid delivery for studies of carotenoid activities and effects on cell models.

biophysics↗

bioRxiv2018

Radioprotective role of cyanobacterial phycobilisomes

It is now generally accepted that cyanobacteria are responsible for production of oxygen, which led to the so-called \"Great Oxygenation Event\". Appearance of dioxygen in Earths atmosphere resulted in formation of the ozone layer and the ionosphere, which caused significant reduction of ionizing radiation levels at the surface of our planet. This event not only increased biological diversity but also canceled the urgency of previously developed mechanisms of DNA protection, which allowed to survive and develop in harsh environmental conditions including exposure to cosmic rays. In order to test the hypothesis if one of the oldest organisms on Earth retained ancient protection mechanisms, we studied the effect of ionizing radiation (IoR, here: -particles with a kinetic energy of about 30 MeV) and space flight during the mission of the Foton-M4 satellite on cells of Synechocystis sp. PCC6803. By analyzing spectral and functional characteristics of photosynthetic membranes we revealed numerous similarities between cells exposed to IoR and after the space mission. In both cases, we found that excitation energy transfer from phycobilisomes to photosystems was interrupted and the concentration of phycobiliproteins was significantly reduced. Although photosynthetic activity was severely suppressed, the effect was reversible and the cells were able to rapidly recover from stress under normal conditions. Moreover, in vitro experiments demonstrated that the effect of IoR on isolated phycobilisomes was completely different from such in vivo. These observations suggest that the actual existence and the uncoupling of phycobilisomes under irradiation stress could play specific role not only in photo-, but also in radioprotection, which was crucial for early stages of evolution and the development of Life on Earth.

biophysics↗

bioRxiv2018

Functional interaction of low-homology FRPs from different cyanobacteria with Synechocystis OCP

Photosynthesis requires a balance between efficient light harvesting and protection against photodamage. The cyanobacterial photoprotection system uniquely relies on the functioning of the photoactive orange carotenoid protein (OCP) that under intense illumination provides fluorescence quenching of the light-harvesting antenna complexes, phycobilisomes. The recently identified fluorescence recovery protein (FRP) binds to the photoactivated OCP and accelerates its relaxation into the basal form, completing the regulatory circle. The molecular mechanism of FRP functioning is largely controversial. Moreover, since the available knowledge has mainly been gained from studying Synechocystis proteins, the cross-species conservation of the FRP mechanism remains unexplored. Besides phylogenetic analysis, we performed a detailed structural-functional analysis of two selected low-homology FRPs by comparing them with Synechocystis FRP (SynFRP). While adopting similar dimeric conformations in solution and preserving binding preferences of SynFRP toward various OCP variants, the low-homology FRPs demonstrated distinct binding stoichiometries and differentially accentuated features of this functional interaction. By providing clues to understand the FRP mechanism universally, our results also establish foundations for upcoming structural investigations necessary to elucidate the FRP-dependent regulatory mechanism.

biochemistry↗

bioRxiv2017

The photocycle of orange carotenoid protein conceals distinct intermediates and asynchronous changes in the carotenoid and protein components

The 35 kDa water-soluble Orange Carotenoid Protein (OCP) is responsible for photoprotection in cyanobacteria. It acts as a light intensity sensor that simultaneously serves as efficient quencher of phycobilisome excitation energy as well as of reactive oxygen species. Photoactivation triggers large-scale conformational rearrangements to convert OCP from the orange OCPO state to the red active signaling state OCPR, as demonstrated by various structural methods. Eventually, such rearrangements imply complete yet reversible separation of structural domains (C- and N-terminal domain) and significant translocation of the carotenoid cofactor. Very recently, dynamic crystallography of OCPO crystals suggested the existence of photocycle intermediates with small-scale rearrangements that may trigger further transitions in the protein. However, the currently existing gap between the ultra-fast picosecond and 100 millisecond time scale of spectroscopic and structural data precludes knowledge about distinct intermediate states. In this study, we took advantage of single 7 ns laser pulses to study carotenoid absorption transients in OCP on the time-scale from 100 ns to 10 s, which allowed us to detect a red intermediate state preceding the red signaling state OCPR. In addition, time-resolved fluorescence spectroscopy and following assignment of carotenoid-induced quenching of different tryptophan residues revealed a novel orange intermediate state, which appears during back-relaxation of photoactivated OCPR to OCPO. Our results show asynchronous changes in the carotenoid and protein components and provide refined mechanistic information about the OCP photocycle as well as introduce new kinetic signatures for future studies of OCP photoactivity and photoprotection.\n\nSignificance statementCyanobacteria utilize the Orange Carotenoid Protein (OCP) to protect their photosynthetic apparatus from the harmful effects of intense sunlight. OCP is a blue light-triggered photoswitch, which undergoes photoconversion from its dark adapted orange to the active red state, the latter being able to interact with the phycobilisome antennae and quench their fluorescence, thus avoiding excessive energy flow to the photosystems. With the help of the fluorescence recovery protein (FRP), OCP detaches from phycobilisomes and can return faster into the orange state. Until now, only the thermodynamically stable orange state and the metastable red state are established in a primitive photocycle. In this work, we apply transient absorption and fluorescence spectroscopy and identify two novel photocycle intermediates of physiological relevance.

biophysics↗

bioRxiv2017

Disruption Of Ankyrin B And Caveolin-1 Interaction Sites Alters Na+,K+-ATPase Lateral Diffusion In HEK293 Cell Plasma Membranes

The Na+,K+-ATPase is a plasma membrane ion transporter of high physiological importance for ion homeostasis and cellular excitability in electrically active tissues. Mutations in the genes coding for Na+,K+-ATPase -subunit isoforms lead to severe human pathologies including Familial Hemiplegic Migraine type 2 (FHM2), Alternating Hemiplegia of Childhood (AHC), Rapid Dystonia Parkinsonism (RDP) or epilepsy. Many of the reported mutations lead to change- or loss-of-function effects, whereas others do not alter the functional properties, but lead to e.g. reduced protein stability, reduced protein expression or defective plasma membrane targeting. Na+,K+-ATPase frequently assembles with other membrane transporters or cellular matrix proteins in specialized plasma membrane microdomains, but the effects of these interactions on targeting or protein mobility are elusive so far. Mutational disruption of established interaction motifs of the Na+,K+-ATPase with ankyrin B and caveolin-1 are expected to result in changes in plasma membrane targeting, changes of the localization pattern, and of the diffusion behavior of the enzyme. We studied the consequences of mutations in these binding sites by monitoring diffusion of eGFP-labeled Na+,K+-ATPase constructs in the plasma membrane of living HEK293T cells by fluorescence correlation spectroscopy (FCS) as well as fluorescence recovery after photobleaching (FRAP) or photoswitching (FRAS) and observed significant differences compared to the wild-type enzyme, with synergistic effects for combinations of interaction site mutations. These measurements expand the possibilities to study the consequences of Na+,K+-ATPase mutations and provide information about the interaction of Na+,K+-ATPase 2-isoform with cellular matrix proteins, the cytoskeleton or other membrane protein complexes.

biophysics↗

bioRxiv2017

The Unique Protein-To-Protein Carotenoid Transfer Mechanism

List of abbreviations\n\nAbstractOrange Carotenoid Protein (OCP) is known to be an effector and regulator of cyanobacterial photoprotection. This 35 kDa water-soluble protein provides specific environment for keto-carotenoids, the excitation of which induced by the absorption of blue-green light causes dramatic but fully reversible rearrangements of the OCP structure, including carotenoid translocation and separation of C- and N-terminal domains upon transition from the basic orange to photoactivated red OCP form. While recent studies significantly improved our understanding of the OCP photocycle and interaction with phycobilisomes and the fluorescence recovery protein, the mechanism of OCP assembly remains unclear. Apparently, this process requires targeted delivery and incorporation of a highly hydrophobic carotenoid molecule into the water-soluble apoprotein of OCP. Recently, we introduced a novel carotenoid carrier protein, COCP, which consists of dimerized C-domain(s) of OCP and can combine with the isolated N-domain to form transient OCP-like species. Here, we demonstrate that in vitro COCP efficiently transfers otherwise tightly bound carotenoid to the full-length OCP apoprotein, resulting in formation of the photoactive OCP from completely photoinactive species. We accurately analyze peculiarities of this carotenoid transfer process which, to the best of our knowledge, seems unique, previously uncharacterized protein-to-protein carotenoid transfer process. We hypothesize that a similar OCP assembly can occur in vivo, substantiating specific roles of the COCP carotenoid carrier in cyanobacterial photoprotection.

biophysics↗

bioRxiv2017

Deletion of the short N-terminal extension in OCP reveals the main site for the FRP binding

The photoactive Orange Carotenoid Protein (OCP) plays a central role in cyanobacterial photoprotection. Photoconversion entails significant structural rearrangements in OCP required for its binding to the phycobilisome to induce excitation energy dissipation, whereas the fluorescence recovery protein (FRP) is required for OCP detachment and restoration of phycobilisome fluorescence. Although key to understanding the whole reversible mechanism of photoprotection, the FRP binding site on OCP has been representing challenge since the discovery of FRP in 2010 and is currently unknown. OCP comprises two structural domains organized into a compact basic orange form due to specific protein-chromophore and inter-domain protein-protein interactions and interacts with FRP tightly only when photoactivated. As an important stabilizing element in the orange OCP, the short A-helix within the N-terminal extension (NTE) binds to OCPs C-terminal domain (CTD), but unfolds upon photoactivation and interferes with phycobilisome binding. By using an alloy of biochemical and biophysical techniques, here we demonstrate that the NTE shares specific structural and functional similarities with FRP and discover the main site of OCP-FRP interactions in the OCP-CTD.

biophysics↗