Dichromic Allophycocyanin Trimer Covering a Broad Spectral Range (550-660†nm).

Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, are a resource for photosynthetic, photonic and fluorescence labeling elements. They cover an exceptionally broad spectral range, but the complex superstructure and assembly have been an obstacle. By replacing in Synechocystis sp. PCC 6803 the biliverdin reductases, we studied the role of chromophores in the assembly of the phycobilisome core. Introduction of the green-absorbing phycoerythrobilin instead of the red-absorbing phycocyanobilin inhibited aggregation. A novel, trimeric allophycocyanin (Dic-APC) was obtained. In the small (110 kDa) unit, the two chromophores, phycoerythrobilin and phytochromobilin, cover a wide spectral range (550 to 660 nm). Due to efficient energy transfer, it provides an efficient artificial light-harvesting element. Dic-APC was generated in vitro by using the contained core-linker, LC , for template-assisted purification and assembly. Labeling the linker provides a method for targeting Dic-APC.

The phycobilisome core-membrane linkers from Synechocystis sp. PCC 6803 and red-algae assemble in the same topology.

The phycobilisomes (PBSs) of cyanobacteria and red-algae are unique megadaltons light-harvesting protein-pigment complexes that utilize bilin derivatives for light absorption and energy transfer. Recently, the high-resolution molecular structures of red-algal PBSs revealed how the multi-domain core-membrane linker (LCM ) specifically organizes the allophycocyanin subunits in the PBS's core. But, the topology of LCM in these structures was different than that suggested for cyanobacterial PBSs based on lower-resolution structures. Particularly, the model for cyanobacteria assumed that the Arm2 domain of LCM connects the two basal allophycocyanin cylinders, whereas the red-algal PBS structures revealed that Arm2 is partly buried in the core of one basal cylinder and connects it to the top cylinder. Here, we show by biochemical analysis of mutations in the apcE gene that encodes LCM , that the cyanobacterial and red-algal LCM topologies are actually the same. We found that removing the top cylinder linker domain in LCM splits the PBS core longitudinally into two separate basal cylinders. Deleting either all or part of the helix-loop-helix domain at the N-terminal end of Arm2, disassembled the basal cylinders and resulted in degradation of the part containing the terminal emitter, ApcD. Deleting the following 30 amino-acids loop severely affected the assembly of the basal cylinders, but further deletion of the amino-acids at the C-terminal half of Arm2 had only minor effects on this assembly. Altogether, the biochemical data are consistent with the red-algal LCM topology, suggesting that the PBS cores in cyanobacteria and red-algae assemble in the same way.

The role of lyases, NblA and NblB proteins and bilin chromophore transfer in restructuring the cyanobacterial light-harvesting complex‡.

Phycobilisomes are large light-harvesting complexes attached to the stromal side of thylakoids in cyanobacteria and red algae. They can be remodeled or degraded in response to changing light and nutritional status. Both the core and the peripheral rods of phycobilisomes contain biliproteins. During biliprotein biosynthesis, open-chain tetrapyrrole chromophores are attached covalently to the apoproteins by dedicated lyases. Another set of non-bleaching (Nb) proteins has been implicated in phycobilisome degradation, among them NblA and NblB. We report in vitro experiments with lyases, biliproteins and NblA/B which imply that the situation is more complex than currently discussed: lyases can also detach the chromophores and NblA and NblB can modulate lyase-catalyzed binding and detachment of chromophores in a complex fashion. We show: (i) NblA and NblB can interfere with chromophorylation as well as chromophore detachment of phycobiliprotein, they are generally inhibitors but in some cases enhance the reaction; (ii) NblA and NblB promote dissociation of whole phycobilisomes, cores and, in particular, allophycocyanin trimers; (iii) while NblA and NblB do not interact with each other, both interact with lyases, apo- and holo-biliproteins; (iv) they promote synergistically the lyase-catalyzed chromophorylation of the ß-subunit of the major rod component, CPC; and (v) they modulate lyase-catalyzed and lyase-independent chromophore transfers among biliproteins, with the core protein, ApcF, the rod protein, CpcA, and sensory biliproteins (phytochromes, cyanobacteriochromes) acting as potential traps. The results indicate that NblA/B can cooperate with lyases in remodeling the phycobilisomes to balance the metabolic requirements of acclimating their light-harvesting capacity without straining the overall metabolic economy of the cell.

Optimization of expression of orange carotenoid protein in Escherichia coli.

Naturally-occurring orange carotenoid protein (OCP) is synthesized in cyanobacteria and red algae for photoprotection. Holo-OCP can be produced with three plasmids in E. coli, which needs two inducers (arabinose and isopropyl ß-D-thiogalactoside) to initiate two processes: one for generation of carotenoid and the other for generation of apo-OCP, so takes about two days. Afterwards, a two-plasmid method using two plasmids in E. coli is established, in which E. coli cells are induced only by isopropyl ß-D-thiogalactoside, so can yield different holo-OCPs from several cyanobacteria within three days. In this work, we optimized the two-plasmid method as follows: (1) re-organization of the two plasmids, letting carotenoid-generating gene, crtW, be arranged together with apo-OCP-generating gene, ocp, in a single plasmid, which causes that both carotenoid and apo-protein were properly produced, (2) modification of several amino acids at the N-terminus of apo-OCP, in this way increasing the yield and purity of holo-OCP. After these optimizations, we can generate much more amount of holo-OCP within shorter time of only 16 h, and pure holo-OCP be conveniently prepared after routine purification. Comparing with the reported data, the general yield of holo-OCP is increased by ∼10-fold under similar conditions. The high quality of the prepared holo-OCPs is verified by fluorescence quenching of the phycobilisomes.

Steered molecular dynamic simulations of conformational lock of Cu, Zn-superoxide dismutase.

The conformational lock was a bio-thermodynamic theory to explain the characteristics of interfaces in oligomeric enzymes and their effects on catalytic activity. The previous studies on superoxide dismutases (Cu, Zn-SODs) showed that the dimeric structure contributed to the high catalytic efficiency and the stability. In this study, steered molecular dynamics simulations were used firstly to study the main interactions between two subunits of Cu, Zn-SODs. The decomposition process study showed that there were not only four pairs of hydrogen bonds but also twenty-five residue pairs participating hydrophobic interactions between A and B chains of SOD, and van der Waals interactions occupied a dominant position among these residue pairs. Moreover, the residue pairs of hydrogen bonds played a major role in maintaining the protein conformation. The analysis of the energy and conformational changes in the SMD simulation showed that there were two groups (two conformational locks) between A and B chains of SOD. The first group consisted of one hydrogen-bond residues pair and seven hydrophobic interactions residues pairs with a total average energy of -30.10 KJ/mol, and the second group of three hydrogen-bond residues pair and eighteen hydrophobic interactions residues pairs formed with a total average energy of -115.23 KJ/mol.

Glucose Oxidase Immobilized on a Functional Polymer Modified Glassy Carbon Electrode and Its Molecule Recognition of Glucose.

In the present study, a glucose oxidase (GluOx) direct electron transfer was realized on an aminated polyethylene glycol (mPEG), carboxylic acid functionalized multi-walled carbon nanotubes (fMWCNTs), and ionic liquid (IL) composite functional polymer modified glassy carbon electrode (GCE). The amino groups in PEG, carboxyl groups in multi-walled carbon nanotubes, and IL may have a better synergistic effect, thus more effectively adjust the hydrophobicity, stability, conductivity, and biocompatibility of the composite functional polymer film. The composite polymer membranes were characterized by cyclic voltammetry (CV), ultraviolet-visible (UV-Vis) spectrophotometer, fluorescence spectroscopy, electrochemical impedance spectroscopy (EIS), and transmission electron microscopy (TEM), respectively. In 50 mM, pH 7.0 phosphate buffer solution, the formal potential and heterogeneous electron transfer constant (ks) of GluOx on the composite functional polymer modified GCE were -0.27 V and 6.5 s-1, respectively. The modified electrode could recognize and detect glucose linearly in the range of 20 to 950 µM with a detection limit of 0.2 µM. The apparent Michaelis-Menten constant (Kmapp) of the modified electrode was 143 µM. The IL/mPEG-fMWCNTs functional polymer could preserve the conformational structure and catalytic activity of GluOx and lead to high sensitivity, stability, and selectivity of the biosensors for glucose recognition and detection.