Residue alterations within a conserved hydrophobic pocket influence light, oxygen, voltage photoreceptor dark recovery.

Light, oxygen, voltage (LOV) photoreceptors are widely distributed throughout all kingdoms of life, and have in recent years, due to their modular nature, been broadly used as sensor domains for the construction of optogenetic tools. For understanding photoreceptor function as well as for optogenetic tool design and fine-tuning, a detailed knowledge of the photophysics, photochemistry, and structural changes underlying the LOV signaling paradigm is instrumental. Mutations that alter the lifetime of the photo-adduct signaling state represent a convenient handle to tune LOV sensor on/off kinetics and, thus, steady-state on/off equilibria of the photoreceptor (or optogenetic switch). Such mutations, however, should ideally only influence sensor kinetics, while being benign with regard to the nature of the structural changes that are induced by illumination, i.e., they should not result in a disruption of signal transduction. In the present study, we identify a conserved hydrophobic pocket for which mutations have a strong impact on the adduct-state lifetime across different LOV photoreceptor families. Using the slow cycling bacterial short LOV photoreceptor PpSB1-LOV, we show that the I48T mutation within this pocket, which accelerates adduct rupture, is otherwise structurally and mechanistically benign, i.e., light-induced structural changes, as probed by NMR spectroscopy and X-ray crystallography, are not altered in the variant. Additional mutations within the pocket of PpSB1-LOV and the introduction of homologous mutations in the LOV photoreceptor YtvA of Bacillus subtilis and the Avena sativa LOV2 domain result in similarly altered kinetics. Given the conserved nature of the corresponding structural region, the here identified mutations should find application in dark-recovery tuning of optogenetic tools and LOV photoreceptors, alike.

Development and Characterization of Flavin-Binding Fluorescent Proteins, Part II: Advanced Characterization.

Flavin-based fluorescent proteins (FbFPs), a class of small fluorescent proteins derived from light-oxygen-voltage (LOV) domains, bind ubiquitous endogenous flavins as chromophores. Due to their unique properties, they can be used as versatile in vivo reporter proteins under aerobic and anaerobic conditions. This chapter presents methodologies for in-depth characterization of the biochemical, spectroscopic, photophysical, and photochemical properties of FbFPs.

Real-Time Tracking of Proton Transfer from the Reactive Cysteine to the Flavin Chromophore of a Photosensing Light Oxygen Voltage Protein.

LOV (light oxygen voltage) proteins are photosensors ubiquitous to all domains of life. A variant of the short LOV protein from Dinoroseobacter shibae (DsLOV) exhibits an exceptionally fast photocycle. We performed time-resolved molecular spectroscopy on DsLOV-M49S and characterized the formation of the thio-adduct state with a covalent bond between the reactive cysteine (C72) and C4a of the FMN. By use of a tunable quantum cascade laser, the weak absorption change of the vibrational band of S-H stretching vibration of C57 was resolved with a time resolution of 10 ns. Deprotonation of C72 proceeded with a time constant of 12 µs which tallies the rise of the thio-adduct state. These results provide valuable information for the mechanistic interpretation of light-induced structural changes in LOV domains, which involves the choreographed sequence of proton transfers, changes in electron density distributions, spin alterations of the latter, and transient bond formation and breakage. Such molecular insight will help develop new optogenetic tools based on flavin photoreceptors.

Complex Evolution of Light-Dependent Protochlorophyllide Oxidoreductases in Aerobic Anoxygenic Phototrophs: Origin, Phylogeny, and Function.

Light-dependent protochlorophyllide oxidoreductase (LPOR) and dark-operative protochlorophyllide oxidoreductase are evolutionary and structurally distinct enzymes that are essential for the synthesis of (bacterio)chlorophyll, the primary pigment needed for both anoxygenic and oxygenic photosynthesis. In contrast to the long-held hypothesis that LPORs are only present in oxygenic phototrophs, we recently identified a functional LPOR in the aerobic anoxygenic phototrophic bacterium (AAPB) Dinoroseobacter shibae and attributed its presence to a single horizontal gene transfer event from cyanobacteria. Here, we provide evidence for the more widespread presence of genuine LPOR enzymes in AAPBs. An exhaustive bioinformatics search identified 36 putative LPORs outside of oxygenic phototrophic bacteria (cyanobacteria) with the majority being AAPBs. Using in vitro and in vivo assays, we show that the large majority of the tested AAPB enzymes are genuine LPORs. Solution structural analyses, performed for two of the AAPB LPORs, revealed a globally conserved structure when compared with a well-characterized cyanobacterial LPOR. Phylogenetic analyses suggest that LPORs were transferred not only from cyanobacteria but also subsequently between proteobacteria and from proteobacteria to Gemmatimonadetes. Our study thus provides another interesting example for the complex evolutionary processes that govern the evolution of bacteria, involving multiple horizontal gene transfer events that likely occurred at different time points and involved different donors.

A Novel Polyester Hydrolase From the Marine Bacterium Pseudomonas aestusnigri - Structural and Functional Insights.

Biodegradation of synthetic polymers, in particular polyethylene terephthalate (PET), is of great importance, since environmental pollution with PET and other plastics has become a severe global problem. Here, we report on the polyester degrading ability of a novel carboxylic ester hydrolase identified in the genome of the marine hydrocarbonoclastic bacterium Pseudomonas aestusnigri VGXO14 T . The enzyme, designated PE-H, belongs to the type IIa family of PET hydrolytic enzymes as indicated by amino acid sequence homology. It was produced in Escherichia coli, purified and its crystal structure was solved at 1.09 Å resolution representing the first structure of a type IIa PET hydrolytic enzyme. The structure shows a typical α/ß-hydrolase fold and high structural homology to known polyester hydrolases. PET hydrolysis was detected at 30°C with amorphous PET film (PETa), but not with PET film from a commercial PET bottle (PETb). A rational mutagenesis study to improve the PET degrading potential of PE-H yielded variant PE-H (Y250S) which showed improved activity, ultimately also allowing the hydrolysis of PETb. The crystal structure of this variant solved at 1.35 Å resolution allowed to rationalize the improvement of enzymatic activity. A PET oligomer binding model was proposed by molecular docking computations. Our results indicate a significant potential of the marine bacterium P. aestusnigri for PET degradation.

Ternary Complex Formation and Photoactivation of a Photoenzyme Results in Altered Protein Dynamics.

The interplay between protein dynamics and catalysis remains a fundamental question in enzymology. We here investigate the ns-timescale dynamics of a light-dependent NADPH:protochlorophyllide oxidoreductase (LPOR), a photoenzyme crucial for chlorophyll synthesis. LPORs catalyze the light-triggered trans addition of a hydride and a proton across the C17═C18 double bond of the chlorophyll precursor protochlorophyllide (Pchlide). Because of the lack of an LPOR structure, the global structural and dynamic consequences of LPOR/Pchlide/NADPH ternary complex formation remain elusive. Moreover, photoactivation of LPORs by low-light preillumination is controversially discussed as unequivocal proof for this phenomenon is lacking. By employing quasielastic neutron spectroscopy (QENS), we show that the formation of the ternary holoprotein complex as well as photoactivation lead to progressive rigidification of the protein. These findings are supported by thermostability measurements, which reveal different melting behavior and thermostabilities for the apo- and holoprotein ternary complexes. Molecular dynamics simulations in good agreement with the experimental QENS results suggest that the increased flexibility observed for the apoprotein stems from structural fluctuations of the NADPH and Pchlide substrate binding sites of the enzyme. On the basis of our results, in conjunction with activity and stability measurements, we provide independent proof for LPOR photoactivation, defined as a process that modifies the protein structure and dynamics, resulting in an increased substrate turnover. Our findings advance the structural and dynamic understanding of LPORs and provide a first link between protein dynamics and catalysis for this enzyme class.

Photoactivation Reduces Side-Chain Dynamics of a LOV Photoreceptor.

We used neutron-scattering experiments to probe the conformational dynamics of the light, oxygen, voltage (LOV) photoreceptor PpSB1-LOV from Pseudomonas putida in both the dark and light states. Global protein diffusion and internal macromolecular dynamics were measured using incoherent neutron time-of-flight and backscattering spectroscopy on the picosecond to nanosecond timescales. Global protein diffusion of PpSB1-LOV is not influenced by photoactivation. Observation-time-dependent global diffusion coefficients were found, which converge on the nanosecond timescale toward diffusion coefficients determined by dynamic light scattering. Mean-square displacements of localized internal motions and effective force constants, , describing the resilience of the proteins were determined on the respective timescales. Photoactivation significantly modifies the flexibility and the resilience of PpSB1-LOV. On the fast, picosecond timescale, small changes in the mean-square displacement and are observed, which are enhanced on the slower, nanosecond timescale. Photoactivation results in a slightly larger resilience of the photoreceptor on the fast, picosecond timescale, whereas in the nanosecond range, a significantly less resilient structure of the light-state protein is observed. For a residue-resolved interpretation of the experimental neutron-scattering data, we analyzed molecular dynamics simulations of the PpSB1-LOV X-ray structure. Based on these data, it is tempting to speculate that light-induced changes in the protein result in altered side-chain mobility mostly for residues on the protruding Jα helix and on the LOV-LOV dimer interface. Our results provide strong experimental evidence that side-chain dynamics play a crucial role in photoactivation and signaling of PpSB1-LOV via modulation of conformational entropy.

Structure and function of a short LOV protein from the marine phototrophic bacterium Dinoroseobacter shibae.

BACKGROUND: Light, oxygen, voltage (LOV) domains are widely distributed in plants, algae, fungi, bacteria, and represent the photo-responsive domains of various blue-light photoreceptor proteins. Their photocycle involves the blue-light triggered adduct formation between the C(4a) atom of a non-covalently bound flavin chromophore and the sulfur atom of a conserved cysteine in the LOV sensor domain. LOV proteins show considerable variation in the structure of N- and C-terminal elements which flank the LOV core domain, as well as in the lifetime of the adduct state. RESULTS: Here, we report the photochemical, structural and functional characterization of DsLOV, a LOV protein from the photoheterotrophic marine α-proteobacterium Dinoroseobacter shibae which exhibits an average adduct state lifetime of 9.6 s at 20°C, and thus represents the fastest reverting bacterial LOV protein reported so far. Mutational analysis in D. shibae revealed a unique role of DsLOV in controlling the induction of photopigment synthesis in the absence of blue-light. The dark state crystal structure of DsLOV determined at 1.5 Å resolution reveals a conserved core domain with an extended N-terminal cap. The dimer interface in the crystal structure forms a unique network of hydrogen bonds involving residues of the N-terminus and the ß-scaffold of the core domain. The structure of photoexcited DsLOV suggests increased flexibility in the N-cap region and a significant shift in the Cα backbone of ß strands in the N- and C-terminal ends of the LOV core domain. CONCLUSIONS: The results presented here cover the characterization of the unusual short LOV protein DsLOV from Dinoroseobacter shibae including its regulatory function, extremely fast dark recovery and an N-terminus mediated dimer interface. Due to its unique photophysical, structural and regulatory properties, DsLOV might thus serve as an alternative model system for studying light perception by LOV proteins and physiological responses in bacteria.

Cofactor trapping, a new method to produce flavin mononucleotide.

We have purified flavin mononucleotide (FMN) from a flavoprotein-overexpressing Escherichia coli strain by cofactor trapping. This approach uses an overexpressed flavoprotein to trap FMN, which is thus removed from the cascade regulating FMN production in E. coli. This, in turn, allows the isolation of highly pure FMN.

High-throughput screening of packed-bed chromatography coupled with SELDI-TOF MS analysis: monoclonal antibodies versus host cell protein.

A feasibility study to couple high throughput screening of packed bed chromatography with mass spectrometric detection by SELDI-TOF MS is presented. As model system monoclonal antibodies (mAb) versus host cell protein (HCP) from an industrial cultivation was chosen. Packed bed chromatography was screened on a TECAN Evo Freedom 200 station using miniaturized chromatographic columns placed on a specially designed array carrier linked to a commercially available T-Stack module. Gradient elution of the bound proteins was performed by applying a multiple step strategy. When analyzing selected HCP peaks as well as the detected antibody peaks throughout the chromatographic runs a direct correlation between applied and detected components was established. The sensitivity of conventional protein A chromatography was found to be lower than SELDI-TOF MS analysis. During initial screening a shift in the elution pattern for one of the monoclonal antibodies detected with all four resins was identified to be a heterogeneity in the mAb glycosylation pattern. In addition, a detailed differentiation between various HCP fractions through out the chromatographic process using SELDI-TOF analysis let to the detection of HCP components possibly adhering to the mAbs during chromatographic separations.