Biology subjects

Moldenhauer, M.

Publications and source records attributed to Moldenhauer, M..

4 recordsLinked to original sources

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 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↗