Charge Carrier Mobility in Poly(N,N'-bis-4-butylphenyl-N,N'-bisphenyl)benzidine Composites with Electron Acceptor Molecules.

Polymer composites based on poly(N,N'-bis-4-butylphenyl-N,N'-bisphenyl)benzidine (poly-TPD) with PCBM and copper(II) pyropheophorbide derivative (Cu-PP) were developed. In thin films of the poly-TPD and Cu-PP composites, the charge carrier mobility was investigated for the first time. In the ternary poly-TPD:PCBM:Cu-PP composite, the electron and hole mobilities are the most balanced compared to binary composites and the photoconductivity is enhanced due to the sensitization by Cu-PP in blue and red spectral ranges. The new composites are promising for use in the development of photodetectors.

Catalytic Mechanism of Fatty Acid Photodecarboxylase: On the Detection and Stability of the Initial Carbonyloxy Radical Intermediate.

In fatty acid photodecarboxylase (FAP), light-induced formation of the primary radical product RCOO⋅ from fatty acid RCOO- occurs in 300 ps, upon which CO2 is released quasi-immediately. Based on the hypothesis that aliphatic RCOO⋅ (spectroscopically uncharacterized because unstable) absorbs in the red similarly to aromatic carbonyloxy radicals such as 2,6-dichlorobenzoyloxy radical (DCB⋅), much longer-lived linear RCOO⋅ has been suggested recently. We performed quantum chemical reaction pathway and spectral calculations. These calculations are in line with the experimental DCB⋅ decarboxylation dynamics and spectral properties and show that in contrast to DCB⋅, aliphatic RCOO⋅ radicals a) decarboxylate with a very low energetic barrier and on the timescale of a few ps and b) exhibit little red absorption. A time-resolved infrared spectroscopy experiment confirms very rapid, ≪300 ps RCOO⋅ decarboxylation in FAP. We argue that this property is required for the observed high quantum yield of hydrocarbons formation by FAP.

Targeted depletion of TRBV9+ T cells as immunotherapy in a patient with ankylosing spondylitis.

Autoimmunity is intrinsically driven by memory T and B cell clones inappropriately targeted at self-antigens. Selective depletion or suppression of self-reactive T cells remains a holy grail of autoimmune therapy, but disease-associated T cell receptors (TCRs) and cognate antigenic epitopes remained elusive. A TRBV9-containing CD8+ TCR motif was recently associated with the pathogenesis of ankylosing spondylitis, psoriatic arthritis and acute anterior uveitis, and cognate HLA-B*27-presented epitopes were identified. Following successful testing in nonhuman primate models, here we report human TRBV9+ T cell elimination in ankylosing spondylitis. The patient achieved remission within 3 months and ceased anti-TNF therapy after 5 years of continuous use. Complete remission has now persisted for 4 years, with three doses of anti-TRBV9 administered per year. We also observed a profound improvement in spinal mobility metrics and the Bath Ankylosing Spondylitis Metrology Index (BASMI). This represents a possibly curative therapy of an autoimmune disease via selective depletion of a TRBV-defined group of T cells. The anti-TRBV9 therapy could potentially be applicable to other HLA-B*27-associated spondyloarthropathies. Such targeted elimination of the underlying cause of the disease without systemic immunosuppression could offer a new generation of safe and efficient therapies for autoimmunity.

Autocatalytic effect boosts the production of medium-chain hydrocarbons by fatty acid photodecarboxylase.

Ongoing climate change is driving the search for renewable and carbon-neutral alternatives to fossil fuels. Photocatalytic conversion of fatty acids to hydrocarbons by fatty acid photodecarboxylase (FAP) represents a promising route to green fuels. However, the alleged low activity of FAP on C2 to C12 fatty acids seemed to preclude the use for synthesis of gasoline-range hydrocarbons. Here, we reveal that Chlorella variabilis FAP (CvFAP) can convert n-octanoic acid in vitro four times faster than n-hexadecanoic acid, its best substrate reported to date. In vivo, this translates into a CvFAP-based production rate over 10-fold higher for n-heptane than for n-pentadecane. Time-resolved spectroscopy and molecular modeling demonstrate that CvFAP's high catalytic activity on n-octanoic acid is, in part, due to an autocatalytic effect of its n-heptane product, which fills the rest of the binding pocket. These results represent an important step toward a bio-based and light-driven production of gasoline-like hydrocarbons.

Photoconduction and Electroluminescence of Copper (II) Protoporphyrin and Chlorin Cu-C-e6.

Cu (II) protoporphyrin Cu-PP-IX and chlorin Cu-C-e6 were found to have both thin solid film formation and charge carrier transport abilities. In the layers deposited by resistive thermal evaporation, the mobilities of holes and electrons are on the order of 10-5 cm2 V-1 s-1. Organic light-emitting diodes incorporating the dye molecules as emitting dopants demonstrate electroluminescence in the UV and near-IR ranges.

Excited-State Properties of Fully Reduced Flavins in Ferredoxin-NADP+ Oxidoreductase.

The fully reduced flavin cofactor (FADred) in ferredoxin-NADP+ oxidoreductase (FNR) is a functional intermediate that displays different catalytic and steady-state spectral properties for enzymes from Bacillus subtilis (BsFNR), Chlorobaculum tepidum (CtFNR), and Rhodopseudomonas palustris (RpFNR). Using ultrafast spectroscopy, we reveal that at physiological pH, photoexcited FADred in BsFNR and RpFNR exhibits unprecedentedly fast decays (dominantly in 6 and 8 ps, respectively), whereas in CtFNR the decay is much slower (∼400 ps), as in other flavoproteins. Correlating these observations with the protonation states of FADred and the dynamic properties of the protein environment, we conclude that the excited state of neutral FADred can be intrinsically short-lived even in proteins, contrasting with the well-documented behavior of the anionic form that systematically displays markedly increased excited-state lifetime upon binding to proteins. This work provides new insight into the photochemistry of fully reduced flavins, which are emerging as functional initial states in bioengineered photocatalysts.

Copper(II) Etioporphyrinate as a Promising Photoluminescent and Electroluminescent Temperature Sensor.

Luminescent temperature sensors are of great interest because they allow remote determination of temperature in transparent media, such as living tissues, as well as on scattering or transparent surfaces of materials. This study analyzes the luminescent properties of copper(II) etioporphyrinate (Cu-EtioP) in a polystyrene film upon variation of temperature from -195 °C to +65 °C in a cryostat. It is shown that the ratio of intensities of phosphorescence transitions in the red spectral region of such a material varies significantly, that is, the material has thermosensory properties. The phosphorescence decay curves of copper(II) etioporphyrinate in a polystyrene film are analyzed. The quantum yield of phosphorescence of copper(II) etioporphyrinate determined by the absolute method was 3.15%. It was also found that the electroluminescence (EL) spectra of copper(II) etioporphyrinate in a poly(9-vinylcarbazole) (PVK) matrix demonstrated a similar change in the spectra in the temperature range -3 °C to +80 °C. That is, copper(II) etioporphyrinate can also be used as a luminescent temperature sensor as part of an active OLED layer.

Parametrization of Force Field Bonded Terms under Structural Inconsistency.

Parametrization of the bonded part of molecular mechanics (MM) force fields (FFs) is typically done by fitting reference quantum mechanical (QM) energies or forces of representative structures. FFs for small molecules are constructed in incremental parametrization procedures, where parameters developed previously are retained for novel molecules, followed by optimization of missing, not previously optimized parameters. Equilibrium QM and MM geometries of molecules can deviate due to parameters transferred from existing molecules in the FF. In this work, we demonstrate that conventional parametrization methods based on fitting QM energies and/or forces to derive parameters for bond and angle terms produce largely suboptimal force constants when MM and QM equilibrium structures deviate. We further developed and tested a new method to derive CHARMM FF parameters based on the potential energy surface scans where a structural deviation between QM and MM optimized geometries is explicitly allowed during parametrization. The test of the new method was performed on a diverse set of 32 molecules. The results show that without any need for additional restraints, the new method produces robust and largely transferable parameters for bond and angle terms. The new method also improves the agreement for the normal modes for all molecules in the test set, reducing the average error in the reproduction of QM normal mode frequencies from 9.5% computed with CGenFF parameters to 6.8% computed with the new parameters. The new method will allow parametrization of molecules under structural deviations, common for force fields for small molecules, producing robust and transferable parameters.

Photochemical and Molecular Dynamics Studies of Halide Binding in Flavoenzyme Glucose Oxidase.

Glucose oxidase (GOX), a characteristic flavoprotein oxidase with widespread industrial applications, binds fluoride (F- ) and chloride (Cl- ). We investigated binding properties of halide inhibitors of GOX through time-resolved spectral characterization of flavin-related photochemical processes and molecular dynamic simulations. Cl- and F- bind differently to the protein active site and have substantial but opposite effects on the population and decay of the flavin excited state. Cl- binds closer to the flavin, whose excited-state decays in <100 fs due to anion-π interactions. Such interactions appear absent in F- binding, which, however, significantly increases the active-site rigidity leading to more homogeneous, picosecond fluorescence decay kinetics. These findings are discussed in relation to the mechanism of halide inhibition of GOX by occupying the accommodation site of catalytic intermediates and increasing the active-site rigidity.

The effect of PbS quantum dots on molecular dynamics and conductivity of PTB7:PC71BM bulk heterojunction as revealed by dielectric spectroscopy.

A ternary photovoltaic blend containing the PTB7 donor component, the PC71BM acceptor component, and colloidal quantum dots of lead sulfide (PbS QDs) was investigated using broadband dielectric spectroscopy. In the dielectric loss spectrum of PTB7:PC71BM:PbS QDs, γ- and ß-relaxation processes in PTB7 were recognized and analyzed in terms of Arrhenius-type equations. To elucidate the effect of PbS QDs on molecular dynamics of PTB7, the activation energies of both processes were evaluated and compared with those obtained for the binary PTB7:PC71BM blend. Using the CELIV method, the charge carrier mobility was estimated. The PbS QD incorporation into the binary blend was shown to decrease both electron and hole mobility in the ternary PTB7:PC71BM:PbS QD blend. For evaluating the charge carrier lifetime in the ternary blend, the Cole-Cole diagrams derived from the dc conductivity data were plotted. The charge carrier lifetime was found to be much less than the hole extraction time, thus providing effective accumulation of charge carries at the electrodes in the ternary blend under investigation.

Copper(II) meso-Tetraphenyl- and meso-Tetrafluorenyl Porphyrinates as Charge Carrier Transporting and Electroluminescent Compounds.

Studies on copper(II) tetrafluorenyl porphyrinate (CuTFP) and copper(II) tetraphenyl porphyrinate (CuTPP) have been focused on the charge carrier transport in their solid films and electroluminescence of their composites. In the dye layers deposited by resistive thermal evaporation, the mobilities of holes and electrons are on the order of 10-5 and 10-6 cm2 V-1 s-1 for the charge transport under the influence of traps, and the charge mobility reaches the order of 10-3 cm2 V-1 s-1 at space-charge-limited current in the nontrapping mode. For the dye molecules, the correlation between the mobility of charge carriers and the distribution of the electron density on the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), which serve as hopping sites for holes and electrons, respectively, is considered. Organic light-emitting diodes incorporating the dye molecules as emitting dopants demonstrate electroluminescence in the near-infrared (IR) range.

Ultrafast photooxidation of protein-bound anionic flavin radicals.

The photophysical properties of anionic semireduced flavin radicals are largely unknown despite their importance in numerous biochemical reactions. Here, we studied the photoproducts of these intrinsically unstable species in five different flavoprotein oxidases where they can be stabilized, including the well-characterized glucose oxidase. Using ultrafast absorption and fluorescence spectroscopy, we unexpectedly found that photoexcitation systematically results in the oxidation of protein-bound anionic flavin radicals on a time scale of less than ∼100 fs. The thus generated photoproducts decay back in the remarkably narrow 10- to 20-ps time range. Based on molecular dynamics and quantum mechanics computations, positively charged active-site histidine and arginine residues are proposed to be the electron acceptor candidates. Altogether, we established that, in addition to the commonly known and extensively studied photoreduction of oxidized flavins in flavoproteins, the reverse process (i.e., the photooxidation of anionic flavin radicals) can also occur. We propose that this process may constitute an excited-state deactivation pathway for protein-bound anionic flavin radicals in general. This hitherto undocumented photochemical reaction in flavoproteins further extends the family of flavin photocycles.

New Unsymmetrically Substituted Benzothiadiazole-Based Luminophores: Synthesis, Optical, Electrochemical Studies, Charge Transport, and Electroluminescent Characteristics.

Three new benzothiadiazole (BTD)-containing luminophores with different configurations of aryl linkers have been prepared via Pd-catalyzed cross-coupling Suzuki and Buchwald-Hartwig reactions. Photophysical and electroluminescent properties of the compounds were investigated to estimate their potential for optoelectronic applications. All synthesized structures have sufficiently high quantum yields in film. The BTD with aryl bridged carbazole unit demonstrated the highest electrons and holes mobility in a series. OLED with light-emitting layer (EML) based on this compound exhibited the highest brightness, as well as current and luminous efficiency. The synthesized compounds are not only luminophores with a high photoluminescence quantum yield, but also active transport centers for charge carriers in EML of OLED devices.

Ion-Driven Self-Assembly of Lanthanide Bis-phthalocyaninates into Conductive Quasi-MOF Nanowires: an Approach toward Easily Recyclable Organic Electronics.

Controlled self-assembly and rapid disintegration of supramolecular nanowires is potentially useful for ecology-friendly organic electronics. Herein, a novel method exploiting the binding between crown-substituted double-decker lanthanide phthalocyaninates (ML2, M = Lu, Ce, Tb) and K+ ions is applied for the one-step fabrication of macroscopically long conductive one-dimensional quasi-metal-organic frameworks. Their properties are controlled by the size of the lanthanide ion guiding the assembly through either intra- or intermolecular interactions. A LuL2 linker with a small interdeck distance yields fully conjugated intermolecular-bonded K+-LuL2 nanowires with a thickness of 10-50 nm, a length of up to 50 µm, and a conductivity of up to 11.4 S cm-1, the highest among them being reported for phthalocyanine assemblies. The large size of CeL2 and TbL2 leads to the formation of mixed intra- and intermolecular K+-ML2 phases with poor electric properties. A field-assisted method is developed to deposit aligned conductive K+-LuL2 assemblies on solids. The solid-supported nanowires can be disintegrated into starting components in a good aprotic solvent for further recycling.

Photochemical processes in flavo-enzymes as a probe for active site dynamics: TrmFO of Thermus thermophilus.

Quenching of flavin fluorescence by electron transfer from neighboring aromatic residues is ubiquitous in flavoproteins. Apart from constituting a functional process in specific light-active systems, time-resolved spectral characterization of the process can more generally be employed as a probe for the active site configuration and dynamics. In the C51A variant of the bacterial RNA-transforming flavoenzyme TrmFO from the bacterium Thermus thermophilus, fluorescence is very short-lived (~ 1 ps), and close-by Tyr343 is known to act as the main quencher, as confirmed here by the very similar dynamics observed in protein variants with modified other potential quenchers, Trp283 and Trp214. When Tyr343 is modified to redox-inactive phenylalanine, slower and highly multiphasic kinetics are observed on the picosecond-nanosecond timescale, reflecting heterogeneous electron donor-acceptor configurations. We demonstrate that Trp214, which is located on a potentially functional flexible loop, contributes to electron donor quenching in this variant. Contrasting with observations in other nucleic acid-transforming enzymes, these kinetics are strikingly temperature-independent. This indicates (a) near-barrierless electron transfer reactions and (b) no exchange between different configurations on the timescale up to at least 2 ns, despite the presumed flexibility of Trp214. Results of extensive molecular dynamics simulations are presented to explain this unexpected finding in terms of slowly exchanging protein configurations.

Additive CHARMM36 Force Field for Nonstandard Amino Acids.

Nonstandard amino acids are both abundant in nature, where they play a key role in various cellular processes, and can be synthesized in laboratories, for example, for the manufacture of a range of pharmaceutical agents. In this work, we have extended the additive all-atom CHARMM36 and CHARMM General force field (CGenFF) to a large set of 333 nonstandard amino acids. These include both amino acids with nonstandard side chains, such as post-translationally modified and artificial amino acids, as well as amino acids with modified backbone groups, such as chromophores composed of several amino acids. Model compounds representative of the nonstandard amino acids were parametrized for protonation states that are likely at the physiological pH of 7 and, for some more common residues, in both d- and l-stereoisomers. Considering all protonation, tautomeric, and stereoisomeric forms, a total of 406 nonstandard amino acids were parametrized. Emphasis was placed on the quality of both intra- and intermolecular parameters. Partial charges were derived using quantum mechanical (QM) data on model compound dipole moments, electrostatic potentials, and interactions with water. Optimization of all intramolecular parameters, including torsion angle parameters, was performed against information from QM adiabatic potential energy surface (PES) scans. Special emphasis was put on the quality of terms corresponding to PES around rotatable dihedral angles. Validation of the force field was based on molecular dynamics simulations of 20 protein complexes containing different nonstandard amino acids. Overall, the presented parameters will allow for computational studies of a wide range of proteins containing nonstandard amino acids, including natural and artificial residues.

Characterization of Light-Induced, Short-Lived Interacting Radicals in the Active Site of Flavoprotein Ferredoxin-NADP+ Oxidoreductase.

Radicals of flavin adenine dinucleotide (FAD), as well as tyrosine and tryptophan, are widely involved as key reactive intermediates during electron-transfer (ET) reactions in flavoproteins. Due to the high reactivity of these species and their corresponding short lifetime, characterization of these intermediates in functional processes of flavoproteins is usually challenging but can be achieved by ultrafast spectroscopic studies of light-activatable flavoproteins. In ferredoxin-NADP+ oxidoreductase from Bacillus subtilis (BsFNR), fluorescence of the FAD cofactor that very closely interacts with a neighboring tyrosine residue (Tyr50) is strongly quenched. Here we study short-lived photoproducts of this enzyme and its variants, with Tyr50 replaced by tryptophan or glycine. Using time-resolved fluorescence and absorption spectroscopies, we show that, upon the excitation of WT BsFNR, ultrafast ET from Tyr50 to the excited FAD cofactor occurs in ∼260 fs, an order of magnitude faster than the decay by charge recombination, facilitating the characterization of the reaction intermediates in the charge-separated state with respect to other recently studied systems. These studies are corroborated by experiments on the Y50W mutant protein, which yield photoproducts qualitatively similar to those observed in other tryptophan-bearing flavoproteins. By combining the experimental results with molecular dynamics simulations and quantum mechanics calculations, we investigate in detail the effects of protein environment and relaxations on the spectral properties of those radical intermediates and demonstrate that the spectral features of radical anionic FAD are highly sensitive to its environment, and in particular to the dynamics and nature of the counterions formed in the photoproducts. Altogether, comprehensive characterizations are provided for important radical intermediates that are generally involved in functional processes of flavoproteins.

Cyclodipeptide Synthases of the NYH Subfamily Recognize tRNA Using an α-Helix Enriched with Positive Residues.

Cyclodipeptide synthases (CDPSs) perform nonribosomal protein synthesis using two aminoacyl-tRNA substrates to produce cyclodipeptides. At present, there are no structural details of the CDPS:tRNA interaction available. Using AlbC, a CDPS that produces cyclo(l-Phe-l-Phe), the interaction between AlbC and its Phe-tRNA substrate was investigated. Simulations of models of the AlbC:tRNA complex, proposed by rigid-body docking or homology modeling, demonstrated that interactions with residues of an AlbC α-helix, α4, significantly contribute to the free energy of binding of AlbC to tRNA. Individual residue contributions to the tRNA binding free energy of the discovered binding mode explain well the available biochemical data, and the results of in vivo assay experiments performed in this work and guided by simulations. In molecular dynamics simulations, the phenylalanyl group predominantly occupied the two positions observed in the experimental structure of AlbC in the dipeptide intermediate state, suggesting that tRNAs of the first and second substrates interact with AlbC in a similar manner. Overall, given the high degree of sequence and structural similarity among the members of the CDPS NYH protein subfamily, the mechanism of the protein:tRNA interaction is expected to be pertinent to a wide range of proteins interacting with tRNA.

Mechanism of Naphthoquinone Selectivity of Thymidylate Synthase ThyX.

Naphthoquinones (NQs) are natural and synthetic compounds with a wide range of biological activities commonly attributed to their redox activity and/or chemical reactivity. However, genetic and biochemical experiments have recently demonstrated that 2-hydroxy-NQs (2-OH-NQs) act as highly specific noncovalent inhibitors of the essential bacterial thymidylate synthase ThyX in a cellular context. We used biochemical experiments and molecular dynamics simulations to elucidate the selective inhibition mechanism of NQ inhibitors of ThyX from Mycobacterium tuberculosis (Mtb). Free energy simulations rationalized how ThyX recognizes the natural substrate dUMP in the N3-ionized form using an arginine, Arg199, in Mtb. The results further demonstrated that 2-OH-NQ, similar to dUMP, binds to ThyX in the ionized form, and the strong and selective binding of 2-OH-NQ to ThyX is also explained by electrostatic interactions with Arg199. The stronger binding of the close analog 5F-dUMP to ThyX and its inhibitory properties compared with dUMP were explained by the stronger acidity of the uracil N3 atom. Our results, therefore, revealed that the ionization of 2-OH-NQs drives their biological activities by mimicking the interactions with the natural substrate. Our observations encourage the rational design of optimized ThyX inhibitors that ultimately may serve as antibiotics.

pKa Calculations with the Polarizable Drude Force Field and Poisson-Boltzmann Solvation Model.

Electronic polarization effects have been suggested to play an important role in proton binding to titratable residues in proteins. In this work, we describe a new computational method for pKa calculations, using Monte Carlo (MC) simulations to sample protein protonation states with the Drude polarizable force field and Poisson-Boltzmann (PB) continuum electrostatic solvent model. While the most populated protonation states at the selected pH, corresponding to residues that are half-protonated at that pH, are sampled using the exact relative free energies computed with Drude particles optimized in the field of the PB implicit solvation model, we introduce an approximation for the protein polarization of low-populated protonation states to reduce the computational cost. The highly populated protonation states used to compute the polarization and pKa's are then iteratively improved until convergence. It is shown that for lysozyme, when considering 9 of the 18 titratable residues, the new method converged within two iterations with computed pKa's differing only by 0.02 pH units from pKa's estimated with the exact approach. Application of the method to predict pKa's of 94 titratable side chains in 8 proteins shows the Drude-PB model to produce physically more correct results as compared to the additive CHARMM36 (C36) force field (FF). With a dielectric constant of two assigned to the protein interior the Root Mean Square (RMS) deviation between computed and experimental pKa's is 2.07 and 3.19 pH units with the Drude and C36 models, respectively, and the RMS deviation using the Drude-PB model is relatively insensitive to the choice of the internal dielectric constant in contrast to the additive C36 model. At the higher internal dielectric constant of 20, pKa's computed with the additive C36 model converge to the results obtained with the Drude polarizable force field, indicating the need to artificially overestimate electrostatic screening in a nonphysical way with the additive FF. In addition, inclusion of both syn and anti orientations of the proton in the neutral state of acidic groups is shown to yield improved agreement with experiment. The present work, which is the first example of the use of a polarizable model for the prediction of pKa's in proteins, shows that the use of a polarizable model represents a more physically correct model for the treatment of electrostatic contributions to pKa shifts in proteins.