Allophycocyanin A is a carbon dioxide receptor in the cyanobacterial phycobilisome.

Light harvesting is fundamental for production of ATP and reducing equivalents for CO2 fixation during photosynthesis. However, electronic energy transfer (EET) through a photosystem can harm the photosynthetic apparatus when not balanced with CO2. Here, we show that CO2 binding to the light-harvesting complex modulates EET in photosynthetic cyanobacteria. More specifically, CO2 binding to the allophycocyanin alpha subunit of the light-harvesting complex regulates EET and its fluorescence quantum yield in the cyanobacterium Synechocystis sp. PCC 6803. CO2 binding decreases the inter-chromophore distance in the allophycocyanin trimer. The result is enhanced EET in vitro and in live cells. Our work identifies a direct target for CO2 in the cyanobacterial light-harvesting apparatus and provides insights into photosynthesis regulation.

Modulated Fluorescence in LB Films Based on DADQs-A Potential Sensing Surface?

Novel fluorescent Langmuir-Blodgett (LB) films have been constructed from three different amphiphilic dicynaoquinodimethanes (DADQs). The DADQs varied in functional group structure, which had an impact on the LB film structure and the fluorescence properties. As the fluorescence of DADQs competes with non-radiative decay (conformational change), the packing and/or free volume in the LB film will influence the average fluorescence lifetime and integrated intensity. The pristine (blank) LB films were then exposed to a selection of non-fluorescent target analytes (some with environmental relevance) and the fluorescence was measured and analyzed relative to the pristine LB film. Exposure of the LB films to selected target analytes results in a modulation of the fluorescence, both with respect to average fluorescence lifetime and integrated intensity. The modulation of the fluorescence is different for different DADQ LB films and can be attributed to restricted non-radiative decays or charge transfer reactions between target analyte and DADQ LB film. The response from the DADQ LB films shows that these systems can be developed into sensing surfaces based on fluorescence measurements.

Applying TADF Emitters in Bioimaging and Sensing-A Novel Approach Using Liposomes for Encapsulation and Cellular Uptake.

A new method for facilitating the delivery, uptake and intracellular localisation of thermally activated delayed fluorescence (TADF) complexes was developed. First, confinement of TADF complexes in liposomes was demonstrated, which were subsequently used as the delivery vehicle for cellular uptake. Confocal fluorescence microscopy showed TADF complexes subsequently localise in the cytoplasm of HepG2 cells. The procedures developed in this work included the removal of molecular oxygen in the liposome preparation without disrupting the liposome structures. Time-resolved fluorescence microscopy (point scanning) showed initial prompt fluorescence followed by a weak, but detectable, delayed fluorescence component for liposomal TADF internalised in HepG2 cells. By demonstrating that it is possible to deliver un-functionalised and/or unshielded TADF complexes, a sensing function for TADFs, such as molecular oxygen, can be envisaged.

A DNA-Binding Bromodomain-Containing Protein Interacts with and Reduces Rx1-Mediated Immune Response to Potato Virus X.

Plant NLR proteins enable the immune system to recognize and respond to pathogen attack. An early consequence of immune activation is transcriptional reprogramming. Some NLRs have been shown to act in the nucleus and interact with transcription factors. The Rx1 NLR protein of potato binds and distorts double-stranded DNA. However, the components of the chromatin-localized Rx1 complex are largely unknown. Here, we report a physical and functional interaction between Rx1 and NbDBCP, a bromodomain-containing chromatin-interacting protein. NbDBCP accumulates in the nucleoplasm and nucleolus, interacts with chromatin, and redistributes Rx1 to the nucleolus in a subpopulation of imaged cells. Rx1 overexpression reduces the interaction between NbDBCP and chromatin. NbDBCP is a negative regulator of Rx1-mediated immune responses to potato virus X (PVX), and this activity requires an intact bromodomain. Previously, Rx1 has been shown to regulate the DNA-binding activity of a Golden2-like transcription factor, NbGlk1. Rx1 and NbDBCP act synergistically to reduce NbGlk1 DNA binding, suggesting a mode of action for NbDBCP's inhibitory effect on immunity. This study provides new mechanistic insight into the mechanism by which a chromatin-localized NLR complex co-ordinates immune signaling after pathogen perception.

TADF Dye-Loaded Nanoparticles for Fluorescence Live-Cell Imaging.

Thermally activated delayed fluorescence (TADF) molecules offer nowadays a powerful tool in the development of novel organic light emitting diodes due to their capability of harvesting energy from non-emissive triplet states without using heavy-metal complexes. TADF emitters have very small energy difference between the singlet and triplet excited states, which makes thermally activated reverse intersystem crossing from the triplet states back to the singlet manifold viable. This mechanism generates a long-lived delayed fluorescence component which can be explored in the sensing of oxygen concentration, local temperature, or used in time-gated optical cell-imaging, to suppress interference from autofluorescence and scattering. Despite this strong potential, until recently the application of TADF outside lighting devices has been hindered due to the low biocompatibility, low aqueous solubility and poor performance in polar media shown by the vast majority of TADF emitters. To achieve TADF luminescence in biological media, careful selection or design of emitters is required. Unfortunately, most TADF molecules are not emissive in polar media, thus complexation with biomolecules or the formation of emissive aggregate states is required, in order to retain the delayed fluorescence that is characteristic of these compounds. Herein, we demonstrate a facile method with great generalization potential that maintains the photophysical properties of solvated dyes by combining luminescent molecules with polymeric nanoparticles. Using an established swelling procedure, two known TADF emitters are loaded onto polystyrene nanoparticles to prepare TADF emitting nanomaterials able to be used in live-cell imaging. The obtained particles were characterized by optical spectroscopy and exhibited the desired TADF emission in aqueous media, due to the polymeric matrix shielding the dye from solvent polarity effects. The prepared nanoparticles were incubated with live human cancer cells and showed very low cytotoxicity and good cellular uptake, thus making fluorescence microscopy imaging possible at low dye concentrations.

Rapid time-resolved Circular Polarization Luminescence (CPL) emission spectroscopy.

Circular polarisation luminescence (CPL) emission spectroscopy is a powerful tool for probing the fundamental chiroptical features of optically emissive chiral molecular systems. However, uptake of CPL spectroscopy has been impeded by the limitations of conventional scanning monochromator (SM) CPL spectrometers, which are costly to acquire and maintain, and typically require tens of minutes to acquire a typical CPL spectrum. Here, we demonstrate a design of CPL spectrometer which uses rapid readout solid state (SS) spectrometer detectors and a dual channel optical layout to acquire CPL spectra in as little as 10 milliseconds. We validate and demonstrate equivalent CPL measurement by measuring CPL spectra of two reference europium(III) complexes. Further, we demonstrate time-gated CPL acquisition, enabling long-lived CPL luminescence to be distinguished from short-lived emission of other fluorescent species. We anticipate that SS-CPL spectrometers will enable flexible, rapid, and relatively low-cost CPL spectroscopy for diverse applications.

A bacteriophage mimic of the bacterial nucleoid-associated protein Fis.

We report the identification and characterization of a bacteriophage λ-encoded protein, NinH. Sequence homology suggests similarity between NinH and Fis, a bacterial nucleoid-associated protein (NAP) involved in numerous DNA topology manipulations, including chromosome condensation, transcriptional regulation and phage site-specific recombination. We find that NinH functions as a homodimer and is able to bind and bend double-stranded DNA in vitro. Furthermore, NinH shows a preference for a 15 bp signature sequence related to the degenerate consensus favored by Fis. Structural studies reinforced the proposed similarity to Fis and supported the identification of residues involved in DNA binding which were demonstrated experimentally. Overexpression of NinH proved toxic and this correlated with its capacity to associate with DNA. NinH is the first example of a phage-encoded Fis-like NAP that likely influences phage excision-integration reactions or bacterial gene expression.

In Vitro and in Cellulo Sensing of Transition Metals Using Time-Resolved Fluorescence Spectroscopy and Microscopy.

In this work we demonstrate that time domain techniques can be used successfully to monitor realtively weak modulations of the fluorescence in sensing applications. The metal sensing complex Newport Green DCF™ can detect selected transition metals in vivo as well as in vitro. Incremental addition of Ni and/or Zn (in vitro) lead to a substantial reduction in the yield of the fast component in a bi-exponential fluorescence decay (τ1 = 150-250 ps) from 60% to 30-35%. This is rationalised as an inhibition of intra-molecular electron transfer in the NPG sensing complex due to metal complexation. In order to explore this effect in cellulo, NIH 3 T3 mouse skin fibroplast cells were pre-incubated with set levels of Ni and Zn, at a constant concentration of NPG. The fluorescence modulation in cellullo was subsequently studied employing both time-resolved fluorescence microscopy and confocal fluorescence microscopy. In correlation with the in vitro observations, similar effects were observed on the fluorescence decay in cellulo.

The intracellular immune receptor Rx1 regulates the DNA-binding activity of a Golden2-like transcription factor.

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable the immune system to recognize and respond to pathogen attack. An early consequence of immune activation is transcriptional reprogramming, and some NLRs have been shown to act in the nucleus and interact with transcription factors. The Rx1 NLR protein of potato is further able to bind and distort double-stranded DNA. However, Rx1 host targets that support a role for Rx1 in transcriptional reprogramming at DNA are unknown. Here, we report a functional interaction between Rx1 and NbGlk1, a Golden2-like transcription factor. Rx1 binds to NbGlk1 in vitro and in planta. NbGlk1 binds to known Golden2-like consensus DNA sequences. Rx1 reduces the binding affinity of NbGlk1 for DNA in vitro. NbGlk1 activates cellular responses to potato virus X, whereas Rx1 associates with NbGlk1 and prevents its assembly on DNA in planta unless activated by PVX. This study provides new mechanistic insight into how an NLR can coordinate an immune signaling response at DNA following pathogen perceptions.

A tight tunable range for Ni(II) sensing and buffering in cells.

The metal affinities of metal-sensing transcriptional regulators co-vary with cellular metal concentrations over more than 12 orders of magnitude. To understand the cause of this relationship, we determined the structure of the Ni(II) sensor InrS and then created cyanobacteria (Synechocystis PCC 6803) in which transcription of genes encoding a Ni(II) exporter and a Ni(II) importer were controlled by InrS variants with weaker Ni(II) affinities. Variant strains were sensitive to elevated nickel and contained more nickel, but the increase was small compared with the change in Ni(II) affinity. All of the variant sensors retained the allosteric mechanism that inhibits DNA binding following metal binding, but a response to nickel in vivo was observed only when the sensitivity was set to respond in a relatively narrow (less than two orders of magnitude) range of nickel concentrations. Thus, the Ni(II) affinity of InrS is attuned to cellular metal concentrations rather than the converse.

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA.

The Potato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor Rx1 Is a Pathogen-dependent DNA-deforming Protein.

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus; however, conserved nuclear targets that support their role in immunity are unknown. Previously, we noted a structural homology between the nucleotide-binding domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC (nucleotide-binding, Apaf-1, R-proteins, and CED-4) domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro, dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger an Rx1-DNA interaction. DNA binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signaling and define DNA as a molecular target of an activated NLR.

Chiral probe development for circularly polarised luminescence: comparative study of structural factors determining the degree of induced CPL with four heptacoordinate europium(III) complexes.

A series of bright, europium(iii) complexes has been prepared based on an achiral heptadentate triazacyclononane ligand bearing two strongly absorbing, coordinated aralkynyl pyridyl moieties. The binding of chiral carboxylates, including α-hydroxy acids such as lactate and mandelate, has been monitored by emission spectroscopy and is signalled by the switching on of strong circularly polarised emission. In each case, an R-chiral carboxylate gave rise to emission typical of a Δ complex, most clearly shown in the form of the ΔJ = 4 transition manifold around 700 nm. Variations in the sign and magnitude of the CPL allow the enantiomeric purity and absolute configuration of the acid to be assessed in a sample. Analysis of the relative energies of the parent aqua complexes and their stereoisomeric adducts has been aided by lifetime measurements and density functional theory calculations.

Oligo(p-phenyleneethynylene) (OPE) molecular wires: synthesis and length dependence of photoinduced charge transfer in OPEs with triarylamine and diaryloxadiazole end groups.

The systematic synthesis and photophysical, electrochemical and computational studies on an extended series of triphenylamine-[C≡C-1,4-C6H2(OR)2]n-C≡C-diphenyl-1,3,4-oxadiazole dyad molecules (the OR groups are at 2,5-positions of the para-phenylene ring and R = C6H13; n = 0-5, compounds 1, 2, 3, 4 and 5, respectively) are reported. Related molecules with identical end groups, triphenylamine-C≡C-1,4-C6H2(OR)2-C≡C-triphenylamine (R = C6H13; 6) and diphenyl-1,3,4-oxadiazole-[C≡C-C6H2(OR)2]2-C≡C-diphenyl-1,3,4-oxadiazole (R = C6H13; 7) were also studied. These D-B-A 1-5, D-B-D 6 and A-B-A 7 (D = electron donor, B = bridge, A = electron acceptor) systems were synthesized using palladium-catalysed cross-coupling reactions of new p-phenyleneethynylene building blocks. Steady-state emission studies on the dyads 1-5 reveal a complicated behavior of the emission that is strongly medium dependent. In low polarity solvents the emission is characterized by a sharp high-energy peak attributed to fluorescence from a locally excited (LE) state. In more polar environments the LE state is effectively quenched by transfer into an intramolecular charge-transfer (ICT) state. The medium dependence is also observed in the quantum yields (QYs) which are high in cyclohexane and low in acetonitrile, thus also indicating charge-transfer character. Low-temperature emission spectra for 2-5 in dichloromethane and diethyl ether also reveal two distinct excited states, namely the LE state and the conventional ICT state, depending on solvent and temperature. Hybrid DFT calculations for 1-7 establish that the OPE bridge is involved in both frontier orbitals where the bridge character increases as the bridge length increases. Computed TD-DFT data on 1-5 assign the emission maxima in cyclohexane as LE transitions. Each time-resolved emission measurement on 2-7 in cyclohexane and diethyl ether reveals a wavelength dependent bi-exponential decay of the emission with a fast component in the 5-61 ps range on blue detection and a slower approximately 1 ns phase, independent of detection wavelength. The fast component is attributed to LE fluorescence and this emission component is rate limited and quenched by transfer into an ICT state. The fast LE fluorescence component varies systematically with conjugation length for the series of D-B-A dyads 2-5. An attenuation factor ß of 0.15 Å(-1) was determined in accordance with an ICT superexchange mechanism.

Ultrafast dynamics and computational studies on diaminodicyanoquinodimethanes (DADQs).

Three diaminodicyanoquinodimethanes, 4-(R(1)R(2)C)-1-[(NC)2C]-C6H4 (R(1),R(2) = H2N, 1; R(1) = 3,5-Me2-4-OCH4H6N-, R(2) = H2N, 2; R(1) = 3,5-Me2-4-OCH4H6N-, R(2) = 4-Me-C5H9N, 3), were investigated using carbon-13 NMR, steady-state, and ultrafast transient absorption and ultrafast fluorescence spectroscopies to unravel the unusual characteristics of this class of chromophores. Computed (GIAO)B3LYP/6-31G* data for the zwitterions 1-3 using necessary solvation (PCM) models were shown to be in excellent agreement with observed structural and carbon-13 NMR data. The ground-state geometries of 1-3 contain a cationic methine group R(1)R(2)C- twisted from the C6H4 ring and an anionic methine group (NC)2C- in plane with the C6H4 ring in solution and solid state. The (13)C chemical shifts of the peak corresponding to the methine carbon at the (NC)2C- group of 1-3 are observed at 32.5-34.7 ppm, which are some 55 ppm upfield compared with the (13)C chemical shift for the methine carbons in TCNQ, 1,4-[(NC)2C]2-C6H4. The decay of the excited state in diaminodicyanoquinodimethanes is fast and dominated by nonradiative processes on the picosecond time scale, which depends on the viscosity of the medium. The dynamics of the excited-state decay is therefore limited by conformational changes through an intramolecular twisting motion. This twisting motion is hindered by friction, which, in turn, also depends on the functional group size of the system. The dominant nonradiative pathways after excitation are due to twisted excited-state conformers according to TD-DFT computations.

Regiospecific formation and unusual optical properties of 2,5-bis(arylethynyl)rhodacyclopentadienes: a new class of luminescent organometallics.

A series of 2,5-bis(arylethynyl)rhodacyclopentadienes has been prepared by a rare example of regiospecific reductive coupling of 1,4-(p-R-phenyl)-1,3-butadiynes (R=H, Me, OMe, SMe, NMe2, CF3, CO2Me, CN, NO2, -C≡C-(p-C6H4-NHex2), -C≡C-(p-C6H4-CO2Oct)) at [RhX(PMe3)4] (1) (X=-C≡C-SiMe3 (a), -C≡C-(p-C6H4-NMe2) (b), -C≡C-C≡C-(p-C6H4-NPh2) (c) or -C≡C-{p-C6H4-C≡C-(p-C6H4 -N(C6H13)2)} (d) or Me (e)), giving the 2,5-bis(arylethynyl) isomer exclusively. The rhodacyclopentadienes bearing a methyl ligand in the equatorial plane (compound 1 e) have been converted into their chloro analogues by reaction with HCl etherate. The rhodacycles thus obtained are stable to air and moisture in the solid state and the acceptor-substituted compounds are even stable to air and moisture in solution. The photophysical properties of the rhodacyclopentadienes are highly unusual in that they exhibit, exclusively, fluorescence between 500-800 nm from the S1 state, with quantum yields of Φ=0.01-0.18 and short lifetimes (τ=0.45-8.20 ns). The triplet state formation (Φ(ISC) =0.57 for 2 a) is exceptionally slow, occurring on the nanosecond timescale. This is unexpected, because the Rh atom should normally facilitate intersystem crossing within femto- to picoseconds, leading to phosphorescence from the T1 state. This work therefore highlights that in some transition-metal complexes, the heavy atom can play a more subtle role in controlling the photophysical behavior than is commonly appreciated.

Induced circularly polarized luminescence arising from anion or protein binding to racemic emissive lanthanide complexes.

A circularly polarized luminescence (CPL) spectrometer has been built and used to study the binding interaction of lactate and four different proteins with racemic EuIII and TbIII complexes in aqueous solution. Lactate binding gives rise to strong induced CPL spectra, and the observed emission dissymmetry factors vary linearly with enantiomeric composition. Particularly strong induced TbIII CPL also characterizes the binding interaction of alpha-1-acid glycoprotein with a dissociation constant, Kd, of 2.5 µM.

Responsive microsecond-lifetime photoluminescent probes for analysis of protein kinases and their inhibitors.

Responsive ARC-Lum probes were used for measurement of the concentration of active protein kinases (PKs) and determination of affinity of inhibitors of PKs. ARC-Lum probes incorporate thiophene or a selenophene heterocycle and a fluorophore conjugated to the lysine residue in the peptide fragment. In the complex with a PK, ARC-Lum probes emit long-lifetime (microsecond-scale) luminescence at the emission wavelengths of the fluorescent label if the complex is illuminated at the excitation wavelength of the thiophene- or selenophene-containing phosphorescence donors. Bisubstrate ARC-Lum probes bind with sub-nanomolar affinity with several PKs of the AGC group. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Experimental and theoretical studies of the photophysical properties of 2- and 2,7-functionalized pyrene derivatives.

Pyrene derivatives substituted at the 2- and 2,7-positions are shown to display a set of photophysical properties different from those of derivatives substituted at the 1-position. It was found that, in the 2- and 2,7-derivatives, there was little influence on the S(2) ← S(0) excitation, which is described as "pyrene-like", and a strong influence on the S(1) ← S(0) excitation, which is described as "substituent-influenced". In contrast, the 1-substituted derivatives display a strong influence on both the S(1) ← S(0) and the S(2) ← S(0) excitations. These observations are rationalized by considering the nature of the orbitals involved in the transitions. The existence of a nodal plane passing through the 2- and 7-positions, perpendicular to the molecular plane in the HOMO and LUMO of pyrene, largely accounts for the different behavior of derivatives substituted at the 2- and 2,7-positions. Herein, we report the photophysical properties of a series of 2-R-pyrenes {R = C(3)H(6)CO(2)H (1), Bpin (2; pin = OCMe(2)CMe(2)O), OC(3)H(6)CO(2)H (3), O(CH(2))(12)Br (4), C≡CPh (5), C(6)H(4)-4-CO(2)Me (6), C(6)H(4)-4-B(Mes)(2) (7), B(Mes)(2) (8)} and 2,7-R(2)-pyrenes {R = Bpin (9), OH (10), C≡C(TMS) (11), C≡CPh (12), C≡C-C(6)H(4)-4-B(Mes)(2) (13), C≡C-C(6)H(4)-4-NMe(2) (14), C(6)H(4)-4-CO(2)C(8)H(17) (15), N(Ph)-C(6)H(4)-4-OMe (16)} whose syntheses are reported elsewhere. Furthermore, we compare their properties to those of several related 1-R-pyrene derivatives {R = C(3)H(6)CO(2)H (17), Bpin (18), C≡CPh (19), C(6)H(4)-4-B(Mes)(2) (20), B(Mes)(2) (21)}. For all derivatives, modest (0.19) to high (0.93) fluorescence quantum yields were observed. For the 2- and 2,7-derivatives, fluorescence lifetimes exceeding 16 ns were measured, with most being ca. 50-80 ns. The 4-(pyren-2-yl)butyric acid derivative (1) has a long fluorescence lifetime of 622 ns, significantly longer than that of the commercially available 4-(pyren-1-yl)butyric acid (17). In addition to measurements of absorption and emission spectra and fluorescence quantum yields and lifetimes, time-dependent density functional theory calculations using the B3LYP and CAM-B3LYP functionals were also performed. A comparison of experimental and theoretically calculated wavelengths shows that both functionals were able to reproduce the trend in wavelengths observed experimentally.

The time domain in co-stained cell imaging: time-resolved emission imaging microscopy using a protonatable luminescent iridium complex.

The intense luminescence of the new complex Ir(ppy)(2)(pybz) (1) within the cytoplasm of live cells can be discriminated from the fluorescence of an organic stain, solely on the basis of the emission timescale {pybzH = 2-pyridyl-benzimidazole}. The protonated form of 1 displays red-shifted emission, and may be implicated in a superior uptake compared to Ir(ppy)(3).