Biology subjects

Maksimov, E. G.

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

7 recordsLinked to original sources

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↗

bioRxiv2016

Assembly of photoactive Orange Carotenoid Protein from its domains unravels a carotenoid shuttle mechanism

The Orange Carotenoid Protein (OCP) is indispensable for cyanobacterial photoprotection by quenching phycobilisome fluorescence upon photoconversion from the orange OCPO to the red OCPR form. Cyanobacterial genomes frequently harbor, besides genes for Orange Carotenoid Proteins (OCPs), several genes encoding homologs of OCPs N- or C-terminal domains (NTD, CTD). Unlike the well-studied NTD homologs, called Red Carotenoid Proteins (RCPs), the role of CTD homologs remains elusive. We show how OCP can be reassembled from its functional domains. Expression of Synechocystis OCP-CTD in carotenoid-producing Escherichia coli yielded violet-colored proteins, which, upon mixing with the RCP-apoprotein, produced an orange-like photoswitchable form that further photoconverted into a species spectroscopically indistinguishable from RCP, thus demonstrating a unique carotenoid shuttle mechanism. The CTD itself is a novel, dimeric carotenoid-binding protein, which effectively quenches singlet oxygen and interacts with the Fluorescence Recovery Protein, assigning physiological roles to CTD homologs and explaining the evolutionary process of OCP formation.\n\nOne Sentence SummaryThe C-domain of cyanobacterial OCP dimerizes, binds a carotenoid, and delivers it to the N-domain forming photoactive OCP.

biophysics↗