Dmitry V. Zlenko

Fluorescence quenching of the phycobilisome terminal emitter LCM from the cyanobacterium Synechocystis sp. PCC 6803 detected in vivo and in vitro

The fluorescence emission of the phycobilisome (PBS) core-membrane linker protein (L(CM)) can be directly quenched by photoactivated orange carotenoid protein (OCP) at room temperature both in vitro and in vivo, which suggests the crucial role of the OCP-L(CM) interaction in non-photochemical quenching (NPQ) of cyanobacteria. This implication was further supported (i) by low-temperature (77K) fluorescence emission and excitation measurements which showed a specific quenching of the corresponding long-wavelength fluorescence bands which belong to the PBS terminal emitters in the presence of photoactivated OCP, (ii) by systematic investigation of the fluorescence quenching and recovery in wild type and L(CM)-less cells of the model cyanobacterium Synechocystis sp. PCC 6803, and (iii) by the impact of dephosphorylation of isolated PBS on the quenching. The OCP binding site within the PBS and the most probable geometrical arrangement of the OCP-allophycocyanin (APC) complex was determined in silico using the crystal structures of OCP and APC. Geometrically modeled attachment of OCP to the PBS core is not at variance with the OCP-L(CM) interaction. It was concluded that besides being a very central element in the PBS to reaction center excitation energy transfer and PBS assembly, L(CM) also has an essential role in the photoprotective light adaptation processes of cyanobacteria.

Assembly of photoactive orange carotenoid protein from its domains unravels a carotenoid shuttle mechanism

The photoswitchable 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 OCP’s 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 that quenches phycobilisome fluorescence and is spectroscopically indistinguishable from RCP, thus demonstrating a unique carotenoid shuttle mechanism. Spontaneous carotenoid transfer also occurs between canthaxanthin-coordinating OCP-CTD and the OCP apoprotein resulting in formation of photoactive OCP. The OCP-CTD itself is a novel, dimeric carotenoid-binding protein, which can coordinate canthaxanthin and zeaxanthin, effectively quenches singlet oxygen and interacts with the Fluorescence Recovery Protein. These findings assign physiological roles to the multitude of CTD homologs in cyanobacteria and explain the evolutionary process of OCP formation.

Cyanobacterial phycobilisomes and phycobiliproteins

In cyanobacteria, phycobilisomes (PBS) act as antenna of the photosynthetic pigment apparatus. They contain brightly colored phycobiliproteins (PBP) and form giant supramolecular complexes (up to 3000–7000 kDa) containing 200 to 500 phycobilin chromophores covalently bound to the proteins. There are over ten various PBP known, which falls into one of three groups: phycoerythrins, phycocyanins, and allophycocyanins. Hollow disks of PBP trimers and hexamers are arranged into cylinders by colorless linker proteins; the cylinders are then assembled into PBS. Typical semidiscoidal PBS consists of a central core formed by three allophycocyanin cylinders and of six lateral cylinders consisting of other PBP and attached as a fan to the nucleus. The PBS number, size, and pigment composition in cyanobacteria depend on light conditions and other ambient factors. While PBSs have certain advantages compared to other antennae, these pigment-protein complexes require more energy for their biosynthesis than the chlorophyll a/b and chlorophyll a/c proteins of oxygenic photosynthetic organisms.

Role of inter-domain cavity in the attachment of the orange carotenoid protein to the phycobilisome core and to the fluorescence recovery protein

Using molecular modeling and known spatial structure of proteins, we have derived a universal 3D model of the orange carotenoid protein (OCP) and phycobilisome (PBS) interaction in the process of non-photochemical PBS quenching. The characteristic tip of the phycobilin domain of the core-membrane linker polypeptide (LCM) forms the attachment site on the PBS core surface for interaction with the central inter-domain cavity of the OCP molecule. This spatial arrangement has to be the most advantageous one because the LCM, as the major terminal PBS-fluorescence emitter, accumulates energy from the most other phycobiliproteins within the PBS before quenching by OCP. In agreement with the constructed model, in cyanobacteria, the small fluorescence recovery protein is wedged in the OCP’s central cavity, weakening the PBS and OCP interaction. The presence of another one protein, the red carotenoid protein, in some cyanobacterial species, which also can interact with the PBS, also corresponds to this model.

Structural modeling of the phycobilisome core and its association with the photosystems

The phycobilisome (PBS) is a major light-harvesting complex in cyanobacteria and red algae. To obtain the detailed structure of the hemidiscoidal PBS core composed of allophycocyanin (APC) and minor polypeptide components, we analyzed all nine available 3D structures of APCs from different photosynthetic species and found several variants of crystal packing that potentially correspond to PBS core organization. Combination of face-to-face APC trimer crystal packing with back-to-back APC hexamer packing suggests two variants of the tricylindrical PBS core. To choose one of these structures, a computational model of the PBS core complex and photosystem II (PSII) dimer with minimized distance between the terminal PBS emitters and neighboring antenna chlorophylls was built. In the selected model, the distance between two types of pigments does not exceed 37 Å corresponding to the Förster mechanism of energy transfer. We also propose a model of PBS and photosystem I (PSI) monomer interaction showing a possibility of supercomplex formation and direct energy transfer from the PBS to PSI.

Computing the self-diffusion coefficient for TIP4P water

A molecular dynamics study was made for the TIP4P model of liquid water. Thermal dependences of water density and radial distribution functions were calculated for model verification. Different methods were used to calculate the self-diffusion coefficient, and assessed for sensitivity to molecular system size and trajectory length. The Green-Kubo formula deriving the diffusion coefficient from the velocity autocorrelation function is preferable in short MD simulations with a high sampling rate, whereas the Einstein equation for diffusion is the method of choice in long simulations. The latter approach yields more stable and reliable results, especially at very short times and for a small number of molecules, if the diffusion coefficient is estimated not from the limit ratio of mean squared displacement to time, but from the slope of the time plot of mean squared displacement.

Conformational Dynamics of the Single Lipopolysaccharide O-Antigen in Solution.

The O-antigen is the most variable and highly immunogenic part of the lipopolysaccharide molecule that covers the surface of Gram-negative bacteria and makes up the first line of cellular defense. To provide insight into the details of the O-antigen arrangement on the membrane surface, we simulated its behavior in solution by molecular dynamics. We developed the energetically favorable O-antigen conformation by analyzing free-energy distributions for its disaccharide fragments. Starting from this conformation, we simulated the behavior of the O-antigen chain on long timescales. Depending on the force field and temperature, the single molecule can undergo reversible or irreversible coil-to-globule transitions. The mechanism of these transitions is related either to the rotation of the carbohydrate residues around O-glycosidic bonds or to flips of the pyranose rings. We found that the presence of rhamnose in the O-antigen chain crucially increases its conformational mobility.

Phycobilisomes from the mutant cyanobacterium Synechocystis sp. PCC 6803 missing chromophore domain of ApcE

Phycobilisome (PBS) is a giant photosynthetic antenna associated with the thylakoid membranes of cyanobacteria and red algae. PBS consists of two domains: central core and peripheral rods assembled of disc-shaped phycobiliprotein aggregates and linker polypeptides. The study of the PBS architecture is hindered due to the lack of the data on the structure of the large ApcE-linker also called LCM. ApcE participates in the PBS core stabilization, PBS anchoring to the photosynthetic membrane, transfer of the light energy to chlorophyll, and, very probably, the interaction with the orange carotenoid protein (OCP) during the non-photochemical PBS quenching. We have constructed the cyanobacterium Synechocystis sp. PCC 6803 mutant lacking 235 N-terminal amino acids of the chromophorylated PBLCM domain of ApcE. The altered fluorescence characteristics of the mutant PBSs indicate that the energy transfer to the terminal emitters within the mutant PBS is largely disturbed. The PBSs of the mutant become unable to attach to the thylakoid membrane, which correlates with the identified absence of the energy transfer from the PBSs to the photosystem II. At the same time, the energy transfer from the PBS to the photosystem I was registered in the mutant cells and seems to occur due to the small cylindrical CpcG2-PBSs formation in addition to the conventional PBSs. In contrast to the wild type Synechocystis, the OCP-mediated non-photochemical PBS quenching was not registered in the mutant cells. Thus, the PBLCM domain takes part in formation of the OCP binding site in the PBS.

Interaction of the orange carotenoid protein with the phycobilisome core and fluorescence recovery protein

Using methods of molecular modeling, we have derived a universal spatial model of the orange carotenoid protein (OCP) and phycobilisome (PBS) core interaction in process of energy excess dissipation. The protrusion of the phy-cobilin domain (PB) of the core-membrane linker polypeptide (Lcm) forms the interaction site for the OCP central cavity on the PBS core surface. This spatial arrangement has to be the most advantageous one because the LCM, as the major terminal PBS-fluorescence emitter, gathers energy from the other phycobiliproteins within the PBS before quenching by OCP. In agreement with the constructed model, the small fluorescence recovery protein (FRP) also interacts with the OCPs central cavity weakening the PBS and OCP binding.

Energy transfer pathways among phycobilin chromophores and fluorescence emission spectra of the phycobilisome core at 293 and 77 K

Energy transfer pathways between phycobiliproteins chromophores in the phycobilisome (PBS) core of the cyanobacterium Synechocystis sp. PCC 6803 were investigated. The computer 3D model of the PBS core with determination of chromophore to chromophore distance was created. Our kinetic equations based on this model allowed us to describe the relative intensities of the fluorescence emission of the short(peaked at 665 nm) and long-wavelength (peaked at 680 nm) chromophores in the PBS core at low and room temperatures. The difference of emissions of the PBS core at 77 and 293 K are due to the back energy transfer, which is observed at room temperature and is negligible at 77 K.