Effect of Molecular Dynamics and Internal Water Contact on the Photophysical Properties of Red pH-Sensitive Proteins.

The pH dependence of the absorption and (time-resolved) fluorescence of two red-shifted fluorescent proteins, mCardinal and mNeptune, was investigated. Decay-associated spectra were measured following fluorescence excitation at 470 nm in PBS buffer with a pH that ranged from 5.5 to 8.0. The fluorescence of both proteins shows two different decay components. mCardinal exhibits an increase in the long-lived fluorescence component with acidification from 1.34 ns at pH 8.0 to 1.62 ns at pH 5.5. An additional fast decay component with 0.64 ns at pH 8.0 up to 1.1 ns at pH 5.5 was found to be blue-shifted compared to the long-lived component. The fluorescence lifetime of mNeptune is insensitive to pH. DAS of mCardinal were simulated assuming a coupled two-level system to describe the 1S state of the chromophore within two different conformations of the protein. MD simulations were conducted to correlate the experimentally observed pH-induced change in the lifetime in mCardinal with its molecular properties. While the chromophores of both protein variants are stabilized by the same number of hydrogen bonds, it was found that the chromophore in mCardinal exhibits more water contacts compared to mNeptune. In mCardinal, interaction between the chromophore and Glu-145 is reduced as compared to mNeptune, but interaction with Thr-147 which is Ser-147 in mNeptune is stronger in mCardinal. Therefore, the dynamics of the excited-state proton transfer (ESPT) might be different in mCardinal and mNeptune. The pH dependency of ESPT is suggested as a key mechanism for pH sensitivity.

How electron tunneling and uphill excitation energy transfer support photochemistry in Halomicronema hongdechloris.

Halomicronema hongdechloris, the first cyanobacterium reported to produce the red-shifted chlorophyll f (Chl f) upon acclimation to far-red light, demonstrates remarkable adaptability to diverse light conditions. The photosystem II (PS II) of this organism undergoes reversible changes in its Chl f content, ranging from practically zero under white-light culture conditions to a Chl f: Chl a ratio of up to 1:8 when exposed to far-red light (FRL) of 720-730 nm for several days. Our ps time- and wavelength-resolved fluorescence data obtained after excitation of living H. hongdechloris cells indicate that the Soret band of a far-red (FR) chlorophyll involved in charge separation absorbs around 470 nm. At 10 K, the fluorescence decay at 715-720 nm is still fast with a time constant of 165 ps indicating an efficient electron tunneling process. There is efficient excitation energy transfer (EET) from 715-720 nm to 745 nm with the latter resulting from FR Chl f, which mainly functions as light-harvesting pigment upon adaptation to FRL. From there, excitation energy reaches the primary donor in the reaction center of PS II with an energetic uphill EET mechanism inducing charge transfer. The fluorescence data are well explained with a secondary donor PD1 represented by a red-shifted Chl a molecule with characteristic fluorescence around 715 nm and a more red-shifted FR Chl f with fluorescence around 725 nm as primary donor at the ChlD1 or PD2 position.

Tuning the Internal Compartmentation of Single-Chain Nanoparticles as Fluorescent Contrast Agents.

Controlling the internal structures of single-chain nanoparticles (SCNPs) is an important factor for their targeted chemical design and synthesis, especially in view of nanosized compartments presenting different local environments as a main feature to control functionality. We here design SCNPs bearing near-infrared fluorescent dyes embedded in hydrophobic compartments for use as contrast agents in pump-probe photoacoustic (PA) imaging, displaying improved properties by the location of the dye in the hydrophobic particle core. Compartment formation is controlled via single-chain collapse and subsequent crosslinking of an amphiphilic polymer using external crosslinkers in reaction media of adjustable polarity. Different SCNPs with hydrodynamic diameters of 6-12 nm bearing adjustable label densities are synthesized. It is found that the specific conditions for single-chain collapse have a major impact on the formation of the desired core-shell structure, in turn adjusting the internal nanocompartments together with the formation of excitonic dye couples, which in turn increase their fluorescence lifetime and PA signal generation. SCNPs with the dye molecules accumulate at the core also show a nonlinear PA response as a function of pulse energy-a property that can be exploited as a contrast mechanism in molecular PA tomography.

Coordination-Induced Band Gap Reduction in a Metal-Organic Framework.

Herein, we report on the synthesis of a microporous, three-dimensional phosphonate metal-organic framework (MOF) with the composition Cu3 (H5 -MTPPA)2 ⋅ 2 NMP (H8 -MTPPA=methane tetra-p-phenylphosphonic acid and NMP=N-methyl-2-pyrrolidone). This MOF, termed TUB1, has a unique one-dimensional inorganic building unit composed of square planar and distorted trigonal bipyramidal copper atoms. It possesses a (calculated) BET surface area of 766.2 m2 /g after removal of the solvents from the voids. The Tauc plot for TUB1 yields indirect and direct band gaps of 2.4 eV and 2.7 eV, respectively. DFT calculations reveal the existence of two spin-dependent gaps of 2.60 eV and 0.48 eV for the alpha and beta spins, respectively, with the lowest unoccupied crystal orbital for both gaps predominantly residing on the square planar copper atoms. The projected density of states suggests that the presence of the square planar copper atoms reduces the overall band gap of TUB1, as the beta-gap for the trigonal bipyramidal copper atoms is 3.72 eV.

Courses Based on iGEM/BIOMOD Competitions Are the Ideal Format for Research-Based Learning of Xenobiology.

Synthetic biology and especially xenobiology, as emerging new fields of science, have reached an intellectual and experimental maturity that makes them suitable for integration into the university curricula of chemical and biological disciplines. Novel scientific fields that include laboratory work are perfect playgrounds for developing highly motivating research-based teaching modules. We believe that research-based learning enriched by digital tools is the best approach for teaching new emerging essentials of academic education. This is especially true when the scientific field as such is still not canonized with text books and best-practice examples. Our experience shows that iGEM/BIOMOD competitions represent an excellent basis for designing research-based courses in xenobiology. Therefore, we present a report on "iGEM-Synthetic Biology" offered at the Technische Universität Berlin as an example.

Arylphosphonate-Tethered Porphyrins: Fluorescence Silencing Speaks a Metal Language in Living Enterocytes*.

We report the application of a highly versatile and engineerable novel sensor platform to monitor biologically significant and toxic metal ions in live human Caco-2 enterocytes. The extended conjugation between the fluorescent porphyrin core and metal ions through aromatic phenylphosphonic acid tethers generates a unique turn off and turn on fluorescence and, in addition, shifts in absorption and emission spectra for zinc, cobalt, cadmium and mercury. The reported fluorescent probes p-H8 TPPA and m-H8 TPPA can monitor a wide range of metal ion concentrations via fluorescence titration and also via fluorescence decay curves. Cu- and Zn-induced turn off fluorescence can be differentially reversed by the addition of common chelators. Both p-H8 TPPA and m-H8 TPPA readily pass the mammalian cellular membrane due to their amphipathic character as confirmed by confocal microscopic imaging of living enterocytes.

Fluorescent and Water Dispersible Single-Chain Nanoparticles: Core-Shell Structured Compartmentation.

Single-chain nanoparticles (SCNPs) are highly versatile structures resembling proteins, able to function as catalysts or biomedical delivery systems. Based on their synthesis by single-chain collapse into nanoparticular systems, their internal structure is complex, resulting in nanosized domains preformed during the crosslinking process. In this study we present proof of such nanocompartments within SCNPs via a combination of electron paramagnetic resonance (EPR) and fluorescence spectroscopy. A novel strategy to encapsulate labels within these water dispersible SCNPs with hydrodynamic radii of ≈5 nm is presented, based on amphiphilic polymers with additional covalently bound labels, attached via the copper catalyzed azide/alkyne "click" reaction (CuAAC). A detailed profile of the interior of the SCNPs and the labels' microenvironment was obtained via electron paramagnetic resonance (EPR) experiments, followed by an assessment of their photophysical properties.

Red, Orange, Green: Light- and Temperature-Dependent Color Tuning in a Cyanobacteriochrome.

Cyanobacteriochromes (CBCRs) are photoreceptor proteins that photoconvert between two parent states and thereby regulate various biological processes. An intriguing property is their variable ultraviolet-visible (UV-vis) absorption that covers the entire spectral range from the far-red to the near-UV region and thus makes CBCRs promising candidates for optogenetic applications. Here, we have studied Slr1393, a CBCR that photoswitches between red- and green-absorbing states (Pr and Pg, respectively). Using UV-vis absorption, fluorescence, and resonance Raman (RR) spectroscopy, a further orange-absorbing state O600 that is in thermal equilibrium with Pr was identified. The different absorption properties of the three states were attributed to the different lengths of the conjugated π-electron system of the phycocyanobilin chromophore. In agreement with available crystal structures and supported by quantum mechanics/molecular mechanics (QM/MM) calculations, the most extended conjugation holds for Pr whereas it is substantially reduced in Pg. Here, the two outer pyrrole rings D and A are twisted out of the plane defined by inner pyrrole rings B and C. For the O600 state, the comparison of the experimental RR spectra with QM/MM-calculated spectra indicates a partially distorted ZZZssa geometry in which ring A is twisted while ring D and the adjacent methine bridge display essentially the same geometry as Pr. The quantitative analysis of temperature-dependent spectra yields an enthalpy barrier of ∼30 kJ/mol for the transition from Pr to O600. This reaction is associated with the movement of a conserved tryptophan residue from the chromophore binding pocket to a solvent-exposed position.

Photosynthesis supported by a chlorophyll f-dependent, entropy-driven uphill energy transfer in Halomicronema hongdechloris cells adapted to far-red light.

The phototrophic cyanobacterium Halomicronema hongdechloris shows far-red light-induced accumulation of chlorophyll (Chl) f, but the involvement of the pigment in photosynthetic energy harvesting by photosystem (PS) II is controversially discussed. While H. hongdechloris contains negligible amounts of Chl f in white-light culture conditions, the ratio of Chl f to Chl a is reversibly changed up to 1:8 under illumination with far-red light (720-730 nm). We performed UV-Vis absorption spectroscopy, time-integrated and time-resolved fluorescence spectroscopy for the calculation of decay-associated spectra (DAS) to determine excitation energy transfer (EET) processes between photosynthetic pigments in intact H. hongdechloris filaments. In cells grown under white light, highly efficient EET occurs from phycobilisomes (PBSs) to Chl a with an apparent time constant of about 100 ps. Charge separation occurs with a typical apparent time constant of 200-300 ps from Chl a. After 3-4 days of growth under far-red light, robust Chl f content was observed in H. hongdechloris and EET from PBSs reached Chl f efficiently within 200 ps. It is proposed based on mathematical modeling by rate equation systems for EET between the PBSs and PSII and subsequent electron transfer (ET) that charge separation occurs from Chl a and excitation energy is funneled from Chl f to Chl a via an energetically uphill EET driven by entropy, which is effective because the number of Chl a molecules coupled to Chl f is at least eight- to tenfold larger than the corresponding number of Chl f molecules. The long lifetime of Chl f molecules in contact to a tenfold larger pool of Chl a molecules allows Chl f to act as an intermediate energy storage level, from which the Gibbs free energy difference between Chl f and Chl a can be overcome by taking advantage from the favorable ratio of degeneracy coefficients, which formally represents a significant entropy gain in the Eyring formulation of the Arrhenius law. Direct evidence for energetically uphill EET and charge separation in PSII upon excitation of Chl f via anti-Stokes fluorescence in far-red light-adapted H. hongdechloris cells was obtained: Excitation by 720 nm laser light resulted in robust Chl a fluorescence at 680 nm that was distinctly temperature-dependent and, notably, increased upon DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) treatment in far-red light-adapted cells. Thus, rather than serving as an excitation energy trap, Chl f in far-red light-adapted H. hongdechloris cells is directly contributing to oxygenic photosynthesis at PSII.

Vinylene-Linked Covalent Organic Frameworks by Base-Catalyzed Aldol Condensation.

Two 2D covalent organic frameworks (COFs) linked by vinylene (-CH=CH-) groups (V-COF-1 and V-COF-2) are synthesized by exploiting the electron deficient nature of the aromatic s-triazine unit of C3 -symmetric 2,4,6-trimethyl-s-triazine (TMT). The acidic terminal methyl hydrogens of TMT can easily be abstracted by a base, resulting in a stabilized carbanion, which further undergoes aldol condensation with multitopic aryl aldehydes to be reticulated into extended crystalline frameworks (V-COFs). Both V-COF-1 (with terepthalaldehyde (TA)) and V-COF-2 (with 1,3,5-tris(p-formylphenyl)benzene (TFPB)) are polycrystalline and exhibit permanent porosity and BET surface areas of 1341 m2 g-1 and 627 m2 g-1 , respectively. Owing to the close proximity (3.52 Å) of the pre-organized vinylene linkages within adjacent 2D layers stacked in eclipsed fashion, [2+2] photo-cycloadditon in V-COF-1 formed covalent crosslinks between the COF layers.

The impact of the phytochromes on photosynthetic processes.

This review describes the phytochrome system in higher plants and cyanobacteria and its role in regulation of photosynthetic processes and stress protection of the photosynthetic apparatus. A relationship between the content of the different phytochromes, the changes in the ratios of the physiologically active forms of phytochromes to their total pool and the resulting influence on photosynthetic processes is reviewed. The role of the phytochromes in the regulation of the expression of genes encoding key photosynthetic proteins, antioxidant enzymes and other components involved in stress signaling is elucidated.

Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein.

Photoprotection in cyanobacteria relies on the interplay between the orange carotenoid protein (OCP) and the fluorescence recovery protein (FRP) in a process termed non-photochemical quenching, NPQ. Illumination with blue-green light converts OCP from the basic orange state (OCPO) into the red-shifted, active state (OCPR) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCPR is converted to OCPO and dissociates from PBs; however, the mode and site of OCPR/FRP interactions remain elusive. Recently, we have introduced the purple OCPW288A mutant as a competent model for the signaling state OCPR (Sluchanko et al., Biochim Biophys Acta 1858:1-11, 2017). Here, we have utilized fluorescence labeling of OCP at its native cysteine residues to generate fluorescent OCP proteins for fluorescence correlation spectroscopy (FCS). Our results show that OCPW288A has a 1.6(±0.4)-fold larger hydrodynamic radius than OCPO, supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCPO, a substantial compaction of the OCPW288A mutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCPW288A or the OCP apoprotein are formed to be detected by FCS. 1:1 complex formation with a micromolar apparent dissociation constant between OCP apoprotein and FRP was confirmed by size-exclusion chromatography. Beyond the established OCP/FRP interaction underlying NPQ cessation, the OCP apoprotein/FRP interaction suggests a more general role of FRP as a scaffold protein for OCP maturation.

Erratum to: Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein.

In Fig. 1a in the original article, the amino acid side chains were incorrectly labeled in the structure representation of the orange carotenoid protein (OCP). The corrected figure is printed in this erratum.

Disruption of Ankyrin B and Caveolin-1 Interaction Sites Alters Na+,K+-ATPase Membrane Diffusion.

The Na+,K+-ATPase is a plasma membrane ion transporter of high physiological importance for ion homeostasis and cellular excitability in electrically active tissues. Mutations in the genes coding for Na+,K+-ATPase α-subunit isoforms lead to severe human pathologies including Familial Hemiplegic Migraine type 2, Alternating Hemiplegia of Childhood, Rapid-onset Dystonia Parkinsonism, or epilepsy. Many of the reported mutations lead to change- or loss-of-function effects, whereas others do not alter the functional properties, but lead to, e.g., reduced protein stability, reduced protein expression, or defective plasma membrane targeting. Na+,K+-ATPase frequently assembles with other membrane transporters or cellular matrix proteins in specialized plasma membrane microdomains, but the effects of these interactions on targeting or protein mobility are elusive so far. Mutation of established interaction motifs of the Na+,K+-ATPase with ankyrin B and caveolin-1 are expected to result in changes in plasma membrane targeting, changes of the localization pattern, and of the diffusion behavior of the enzyme. We studied the consequences of mutations in these binding sites by monitoring diffusion of eGFP-labeled Na+,K+-ATPase constructs in the plasma membrane of HEK293T cells by fluorescence correlation spectroscopy as well as fluorescence recovery after photobleaching or photoswitching, and observed significant differences compared to the wild-type enzyme, with synergistic effects for combinations of interaction site mutations. These measurements expand the possibilities to study the consequences of Na+,K+-ATPase mutations and provide information about the interaction of Na+,K+-ATPase α-isoforms with cellular matrix proteins, the cytoskeleton, or other membrane protein complexes.

Investigation of the NADH/NAD+ ratio in Ralstonia eutropha using the fluorescence reporter protein Peredox.

Ralstonia eutropha is a hydrogen-oxidizing ("Knallgas") bacterium that can easily switch between heterotrophic and autotrophic metabolism to thrive in aerobic and anaerobic environments. Its versatile metabolism makes R. eutropha an attractive host for biotechnological applications, including H2-driven production of biodegradable polymers and hydrocarbons. H2 oxidation by R. eutropha takes place in the presence of O2 and is mediated by four hydrogenases, which represent ideal model systems for both biohydrogen production and H2 utilization. The so-called soluble hydrogenase (SH) couples reversibly H2 oxidation with the reduction of NAD+ to NADH and has already been applied successfully in vitro and in vivo for cofactor regeneration. Thus, the interaction of the SH with the cellular NADH/NAD+ pool is of major interest. In this work, we applied the fluorescent biosensor Peredox to measure the [NADH]:[NAD+] ratio in R. eutropha cells under different metabolic conditions. The results suggest that the sensor operates close to saturation level, indicating a rather high [NADH]:[NAD+] ratio in aerobically grown R. eutropha cells. Furthermore, we demonstrate that multicomponent analysis of spectrally-resolved fluorescence lifetime data of the Peredox sensor response to different [NADH]:[NAD+] ratios represents a novel and sensitive tool to determine the redox state of cells.

Characterization of Frex as an NADH sensor for in vivo applications in the presence of NAD+ and at various pH values.

The fluorescent biosensor Frex, recently introduced as a sensitive tool to quantify the NADH concentration in living cells, was characterized by time-integrated and time-resolved fluorescence spectroscopy regarding its applicability for in vivo measurements. Based on the purified sensor protein, it is shown that the NADH dependence of Frex fluorescence can be described by a Hill function with a concentration of half-maximal sensor response of K D ≈ 4 µM and a Hill coefficient of n ≈ 2. Increasing concentrations of NADH have moderate effects on the fluorescence lifetime of Frex, which changes by a factor of two from about 500 ps in the absence of NADH to 1 ns under fluorescence-saturating NADH concentrations. Therefore, the observed sevenfold rise of the fluorescence intensity is primarily ascribed to amplitude changes. Notably, the dynamic range of Frex sensitivity towards NADH highly depends on the NAD+ concentration, while the apparent K D for NADH is only slightly affected. We found that NAD+ has a strong inhibitory effect on the fluorescence response of Frex during NADH sensing, with an apparent NAD+ dissociation constant of K I ≈ 400 µM. This finding was supported by fluorescence lifetime measurements, which showed that the addition of NAD+ hardly affects the fluorescence lifetime, but rather reduces the number of Frex molecules that are able to bind NADH. Furthermore, the fluorescence responses of Frex to NADH and NAD+ depend critically on pH and temperature. Thus, for in vivo applications of Frex, temperature and pH need to be strictly controlled or considered during data acquisition and analysis. If all these constraints are properly met, Frex fluorescence intensity measurements can be employed to estimate the minimum NADH concentration present within the cell at sufficiently low NAD+ concentrations below 100 µM.

Excitation energy transfer in phycobiliproteins of the cyanobacterium Acaryochloris marina investigated by spectral hole burning.

The cyanobacterium Acaryochloris marina developed two types of antenna complexes, which contain chlorophyll-d (Chl d) and phycocyanobilin (PCB) as light-harvesting pigment molecules, respectively. The latter membrane-extrinsic complexes are denoted as phycobiliproteins (PBPs). Spectral hole burning was employed to study excitation energy transfer and electron-phonon coupling in PBPs. The data reveal a rich spectral substructure with a total of four low-energy electronic states whose absorption bands peak at 633, 644, 654, and at about 673 nm. The electronic states at ~633 and 644 nm can be tentatively attributed to phycocyanin (PC) and allophycocyanin (APC), respectively. The remaining low-energy electronic states including the terminal emitter at 673 nm may be associated with different isoforms of PC, APC, or the linker protein. Furthermore, the hole burning data reveal a large number of excited state vibrational frequencies, which are characteristic for the chromophore PCB. In summary, the results are in good agreement with the low-energy level structure of PBPs and electron-phonon coupling parameters reported by Gryliuk et al. (BBA 1837:1490-1499, 2014) based on difference fluorescence line-narrowing experiments.

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.

Gernot Renger (1937-2013): his life, Max-Volmer Laboratory, and photosynthesis research.

Gernot Renger (October 23, 1937-January 12, 2013), one of the leading biophysicists in the field of photosynthesis research, studied and worked at the Max-Volmer-Institute (MVI) of the Technische Universität Berlin, Germany, for more than 50 years, and thus witnessed the rise and decline of photosynthesis research at this institute, which at its prime was one of the leading centers in this field. We present a tribute to Gernot Renger's work and life in the context of the history of photosynthesis research of that period, with special focus on the MVI. Gernot will be remembered for his thought-provoking questions and his boundless enthusiasm for science.

The Signaling State of Orange Carotenoid Protein.

Orange carotenoid protein (OCP) is the photoactive protein that is responsible for high light tolerance in cyanobacteria. We studied the kinetics of the OCP photocycle by monitoring changes in its absorption spectrum, intrinsic fluorescence, and fluorescence of the Nile red dye bound to OCP. It was demonstrated that all of these three methods provide the same kinetic parameters of the photocycle, namely, the kinetics of OCP relaxation in darkness was biexponential with a ratio of two components equal to 2:1 independently of temperature. Whereas the changes of the absorption spectrum of OCP characterize the geometry and environment of its chromophore, the intrinsic fluorescence of OCP reveals changes in its tertiary structure, and the fluorescence properties of Nile red indicate the exposure of hydrophobic surface areas of OCP to the solvent following the photocycle. The results of molecular-dynamics studies indicated the presence of two metastable conformations of 3'-hydroxyechinenone, which is consistent with characteristic changes in the Raman spectra. We conclude that rotation of the ß-ionylidene ring in the C-terminal domain of OCP could be one of the first conformational rearrangements that occur during photoactivation. The obtained results suggest that the photoactivated form of OCP represents a molten globule-like state that is characterized by increased mobility of tertiary structure elements and solvent accessibility.