Engineering and application of a biosensor with focused ligand specificity.

Cell factories converting bio-based precursors to chemicals present an attractive avenue to a sustainable economy, yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low-throughput endeavor. For this reason, transcriptional biosensors attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional regulators (TRs) often prohibits the development of such ultra-high-throughput screens. Here, we solve the structure of the TR LysG of Corynebacterium glutamicum, which detects all three basic amino acids. Based on this information, we follow a semi-rational engineering approach using a FACS-based screening/counterscreening strategy to generate an L-lysine insensitive LysG-based biosensor. This biosensor can be used to isolate L-histidine-producing strains by FACS, showing that TR engineering towards a more focused ligand spectrum can expand the scope of application of such metabolite sensors.

Structure-Based Design of Versatile Biosensors for Small Molecules Based on the PAS Domain of a Thermophilic Histidine Kinase.

The development of biosensors for in vitro quantification of small molecules such as metabolites or man-made chemicals is still a major challenge. Here we show that engineered variants of the sensory PAS domain of the histidine kinase CitA of the thermophilic bacterium Geobacillus thermoleovorans represent promising alternatives to established biorecognition elements. By combining binding site grafting and rational design we constructed protein variants binding l-malate, ethylmalonate, or the aromatic compound phthalate instead of the native ligand citrate. Due to more favorable entropy contributions, the wild-type protein and its engineered variants exhibited increased (nano- to micromolar) affinities and improved enantioselectivity compared to CitA homologues of mesophilic organisms. Ligand binding was directly converted into an optical signal that was preserved after immobilization of the protein. A fluorescently labeled variant was used to quantify ethylmalonate, an urinary biomarker for ethylmalonic encephalopathy, in synthetic urine, thereby demonstrating the applicability of the sensor in complex samples.

Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity.

Antibody glycosylation is a key parameter in the optimization of antibody therapeutics. Here, we describe the production of the anti-cancer monoclonal antibody rituximab in the unicellular ciliate, Tetrahymena thermophila. The resulting antibody demonstrated enhanced antibody-dependent cell-mediated cytotoxicity, which we attribute to unusual N-linked glycosylation. Detailed chromatographic and mass spectrometric analysis revealed afucosylated, oligomannose-type glycans, which, as a whole, displayed isomeric structures that deviate from the typical human counterparts, but whose branches were equivalent to fragments of metabolic intermediates observed in human glycoproteins. From the analysis of deposited crystal structures, we predict that the ciliate glycans adopt protein-carbohydrate interactions with the Fc domain that closely mimic those of native complex-type glycans. In addition, terminal glucose structures were identified that match biosynthetic precursors of human glycosylation. Our results suggest that ciliate-based expression systems offer a route to large-scale production of monoclonal antibodies exhibiting glycosylation that imparts enhanced cell killing activity.

Critical Assessment of Protein Cross-Linking and Molecular Docking: An Updated Model for the Interaction Between Photosystem II and Psb27.

Photosystem II (PSII) is a large membrane-protein complex composed of about 20 subunits and various cofactors, which mediates the light-driven oxidation of water and reduction of plastoquinone, and is part of the photosynthetic electron transfer chain that is localized in the thylakoid membrane of cyanobacteria, algae, and plants. The stepwise assembly of PSII is guided and facilitated by numerous auxiliary proteins that play specific roles in this spatiotemporal process. Psb27, a small protein localized in the thylakoid lumen, appears to associate with an intermediate PSII complex that is involved in assembly of the Mn4CaO5 cluster. Its precise binding position on the PSII intermediate remains elusive, as previous approaches to the localization of Psb27 on PSII have yielded contradictory results. This was our motivation for a critical assessment of previously used methods and the development of an improved analysis pipeline. The combination of chemical cross-linking and mass spectrometry (CX-MS) with isotope-coded cross-linkers was refined and validated with reference to the PSII crystal structure. Psb27 was localized on the PSII surface adjacent to the large lumenal domain of CP43 on the basis of a cross-link connecting Psb27-K91 to CP43-K381. Additional contacts associating Psb27 with CP47 and the C-termini of D1 and D2 were detected by surface plasmon resonance (SPR) spectroscopy. This information was used to model the binding of Psb27 to the PSII surface in a region that is occupied by PsbV in the mature complex.

Localization of the CyanoP binding site on photosystem II by surface plasmon resonance spectroscopy.

Photosystem II (PSII), a large multi subunit membrane protein complex localized in the thylakoid membrane of cyanobacteria and chloroplasts, is the only known enzyme that catalyzes the light-driven oxidation of water. In addition to the membrane intrinsic part of PSII, efficient oxygen evolution requires soluble protein subunits at its luminal interface. In contrast to the detailed crystal structure of the active cyanobacterial complex the characterization of intermediate PSII species related to its assembly and repair is hampered by their instability or low abundance. As most structural variations of the corresponding PSII species are based on a different set of protein factors bound to the luminal interface of the complex we developed a system for interaction analysis between PSII and its soluble interaction partners based on surface plasmon resonance (SPR) spectroscopy. The assay was validated by the correct localization of the extrinsic PSII proteins PsbO, PsbV, and PsbU on the luminal PSII surface and used to determine the unknown binding position of CyanoP, the cyanobacterial homolog of higher plant PsbP. The CyanoP binding site was clearly localized in the center of PSII at a position, which is occupied by the PsbO subunit in mature PSII complexes. Consistently, we demonstrate selective binding of CyanoP to an inactive PSII assembly intermediate that lacks the extrinsic subunits PsbO, PsbV, and PsbU. These findings suggest, that CyanoP functions in the dynamic lifecycle of PSII, possibly in the association of CP47 and CP43 or in photoactivation of the oxygen-evolving complex.

Functional characterization of the small regulatory subunit PetP from the cytochrome b6f complex in Thermosynechococcus elongatus.

The cyanobacterial cytochrome b(6)f complex is central for the coordination of photosynthetic and respiratory electron transport and also for the balance between linear and cyclic electron transport. The development of a purification strategy for a highly active dimeric b(6)f complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 enabled characterization of the structural and functional role of the small subunit PetP in this complex. Moreover, the efficient transformability of this strain allowed the generation of a ΔpetP mutant. Analysis on the whole-cell level by growth curves, photosystem II light saturation curves, and P700(+) reduction kinetics indicate a strong decrease in the linear electron transport in the mutant strain versus the wild type, while the cyclic electron transport via photosystem I and cytochrome b(6)f is largely unaffected. This reduction in linear electron transport is accompanied by a strongly decreased stability and activity of the isolated ΔpetP complex in comparison with the dimeric wild-type complex, which binds two PetP subunits. The distinct behavior of linear and cyclic electron transport may suggest the presence of two distinguishable pools of cytochrome b(6)f complexes with different functions that might be correlated with supercomplex formation.

Assembly of the water-oxidizing complex in photosystem II.

The efficient incorporation and assembly of calcium, chloride and manganese followed by photoactivation of the water-oxidizing complex (WOC) is a prerequisite for the unique water-splitting activity of photosystem II. This minireview summarizes the recent results on incorporation and storage of the inorganic cofactors, photoactivation of the WOC and assembly of the protein environment at the donor site of PSII in cyanobacteria with a special focus on the role of the Psb27 protein.

Dynamics of the cyanobacterial photosynthetic network: communication and modification of membrane protein complexes.

Cyanobacterial photosystem 2 and cytochrome b(6)f complexes have been structurally resolved up to the molecular level while the adjustment of their function in response to environmental and intracellular parameters is based on various modifications of these complexes which have not yet been resolved in detail. This minireview summarizes recent results on two central modifications for each complex: (a) for the cytochrome b(6)f complex the implication of PetP, a new subunit, and of three copies of PetC, the Rieske protein, for the fine-tuning of the photosynthetic electron transport is evaluated; (b) for photosystem 2, the heterogeneity of the D1 subunit and the role of subunit Psb27 is discussed in relation to stress response and the biogenesis/repair cycle. The presented "dynamic" models for both complexes should illustrate the need to complement structural by more extensive functional models which consider the flexibility of individual complexes in the physiological context - beyond structure.

Sequence-specific (1)H, (13)C, and (15)N backbone assignment of Psb27 from Synechocystis PCC 6803.

Photosystem II (PSII) is a large membrane protein complex that uses light to split water into molecular oxygen, protons, and electrons. Here we report the (1)H, (15)N and (13)C backbone chemical shift assignments for the Psb27 protein of Photosystem II from Synechocystis PCC 6803. These assignments will now provide the basis for the structural analysis of the Psb27 protein.

Structure of Psb27 in solution: implications for transient binding to photosystem II during biogenesis and repair.

Psb27 is a membrane-extrinsic subunit of photosystem II (PSII) where it is involved in the assembly and maintenance of this large membrane protein complex that catalyzes one of the key reactions in the biosphere, the light-induced oxidation of water. Here, we report for the first time the structure of Psb27 that was not observed in the previous crystal structures of PSII due to its transient binding mode. The Psb27 structure shows that the core of the protein is a right-handed four-helix bundle with an up-down-up-down topology. The electrostatic potential of the surface generated by the amphipathic helices shows a dipolar distribution which fits perfectly to the major PsbO binding site on the PSII complex. Moreover, the presented docking model could explain the function of Psb27, which prevents the binding of PsbO to facilitate the assembly of the Mn(4)Ca cluster.