Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy.

Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule.

Significance of hydrogen bonding networks in the proton-coupled electron transfer reactions of photosystem II from a quantum-mechanics perspective.

The photosynthetic protein complex, photosystem II (PSII), conducts the light-driven water-splitting reaction with unrivaled efficiency. Proton-coupled electron transfer (PCET) reactions at the redox-active tyrosine residues are thought to play a critical role in the water-splitting chemistry. Addressing the fundamental question as to why the tyrosine residue, YZ, is kinetically competent in comparison to a symmetrically placed tyrosine residue, YD, is important for the elucidation of the mechanism of PCET in the water-splitting reaction of PSII. Here, using all-quantum-mechanical calculations we study PCET at the YZ and YD residues of PSII. We find that when YZ is in its protein matrix under physiological conditions, the HOMO of YZ constitutes the HOMO of the whole system. In contrast, the HOMO of YD is buried under the electronic states localized elsewhere in the protein matrix and PCET at YD requires the transfer of the phenolic proton, which elevates the HOMO of YD to become the HOMO of the whole system. This leads to the oxidation of YD, albeit on a slower timescale. Our study reveals that the key differences between the electronic structure of YZ and YD are primarily determined by the protonation state of the respective hydrogen-bonding partners, D1-His190 and D2-His189, or more generally by the H-bonding network of the protein matrix.

Muon disrupts AKT hydrogen bond network in cancer.

A cancer microenvironment generates strong hydrogen bond network system by the positive feedback loops supporting cancer complexity and robustness. Such network functions through the AKT locus generating high entropic energy supporting cancer metastatic robustness. Charged lepton particle muon follows the rule of Bragg effect during a collision with hydrogen network in cancer cells. Muon beam dismantles hydrogen bond network in cancer by the muon-catalyzed fusion, leading to apoptosis of cancer cells. Muon induces cumulative energy appearance on the hydrogen bond network in a cancer cell with its fast decay to an electron and two neutrinos. Thus, muon beam, muonic atom, muon neutrino shower, and electrons simultaneously cause fast neutralization of the AKT hydrogen bond network by the conversion of hydrogen into deuterium or helium, inactivating the hydrogen bond networks and inducing failure of cancer complexity and robustness with the disappearance of a malignant phenotype.

Toward Developing a Predictive Approach To Assess Electron Beam Instability during Transmission Electron Microscopy of Drug Molecules.

During drug development control of polymorphism, particle properties and impurities are critical for ensuring a good quality, reproducible, and safe medicine. A wide variety of analytical techniques are employed in demonstrating the regulators control over the drug substance and product manufacturing, storage, and supply. Transmission electron microscopy (TEM) offers the opportunity to analyze in detail pharmaceutical systems at a length scale and limit of detection not readily achieved by many traditional techniques. However, the use of TEM as a characterization tool for drug development is uncommon due to possible damage caused by the electron beam. This work outlines the development of a model, using molecular descriptors, to predict the electron beam stability of active pharmaceutical ingredients (API). For a given set of conditions and a particular imaging or analytical mode, the total number of electrons per unit area, which causes observable damage to a sample in the TEM, can be defined as the critical fluence ( CF). Here the CF of 20 poorly water-soluble APIs were measured using selected area electron diffraction. Principal component analysis was used to select the most influential molecular descriptors on CF, which were shown to be descriptors involving the degree of conjugation, the number of hydrogen bond donors and acceptors, and the number of rotatable bonds. These were used to generate several multiple linear regression models. The model that provided the best fit to the measured CF data included the ratio of the number of conjugated carbons to nonconjugated carbons, the ratio of the number of hydrogen bond donors to acceptors, and the ratio of the number of hydrogen bond acceptors to donors. Using this model, the CF of the majority of the compounds was predicted within ±2 e-/Å2. Molecules with no hydrogen bond acceptors did not fit the model accurately possibly due to the limited sample size or the influence of other parameters not included in this model, such as intermolecular bond energies. The model presented can be used to support pharmaceutical development by quickly assessing the stability of other poorly soluble drugs in TEM. Provided that the model suggests that the API is relatively stable to electron irradiation, TEM offers the prospect of determining the presence of crystalline material at low levels at length scales and limits of detection unobtainable by other techniques. This is particularly so for amorphous solid dispersions.

A Robust, Self-Healable, and Shape Memory Supramolecular Hydrogel by Multiple Hydrogen Bonding Interactions.

A versatile double-network (DN) hydrogel with two noncovalent crosslinked networks is synthesized by multiple hydrogen bonding (H-bonding) interactions. The DN hydrogels are synthesized via a heating-cooling photopolymerization process by adding all reactants of agar, N-acryloyl glycinamide (NAGA) and N-benzylacrylamide (NBAA) monomers, UV initiators to a single water pot. Poly(N-acryloyl glycinamide-co-N-benzyl acrylamide) (P(NAGA-co-NBAA)) with a triple amide in one side group is synthesized via UV-light polymerization between NAGA and NBAA, forming a strong intermolecular H-bonding network. Meanwhile, the intramolecular H-bonding network is formed between P(NAGA-co-NBAA) and agars. The sol-gel phase transition of agars at 86 °C generates the molecular entanglement network. Such a double network enables the hydrogel high self-healing efficiency (about 95%), good shape memory ability, and high mechanical strength (1.1 MPa). Additionally, the DN hydrogel is completely crosslinked by multiple hydrogen bonds (H-bonds) and the physical crosslinking of agar without extra potential toxic chemical crosslinker. The DN hydrogels find extensive applications in the biomedical materials due to their excellent biocompatibility.

Proton transfer reactions in the red light-activatable channelrhodopsin variant ReaChR and their relevance for its function.

Channelrhodopsins (ChRs) are light-gated ion channels widely used for activating selected cells in large cellular networks. ChR variants with a red-shifted absorption maximum, such as the modified Volvox carteri ChR1 red-activatable channelrhodopsin ("ReaChR," λmax = 527 nm), are of particular interest because longer wavelengths allow optical excitation of cells in deeper layers of organic tissue. In all ChRs investigated so far, proton transfer reactions and hydrogen bond changes are crucial for the formation of the ion-conducting pore and the selectivity for protons versus cations, such as Na+, K+, and Ca2+ (1). By using a combination of electrophysiological measurements and UV-visible and FTIR spectroscopy, we characterized the proton transfer events in the photocycle of ReaChR and describe their relevance for its function. 1) The central gate residue Glu130 (Glu90 in Chlamydomonas reinhardtii (Cr) ChR2) (i) undergoes a hydrogen bond change in D → K transition and (ii) deprotonates in K → M transition. Its negative charge in the open state is decisive for proton selectivity. 2) The counter-ion Asp293 (Asp253 in CrChR2) receives the retinal Schiff base proton during M-state formation. Starting from M, a photocycle branching occurs involving (i) a direct M → D transition and (ii) formation of late photointermediates N and O. 3) The DC pair residue Asp196 (Asp156 in CrChR2) deprotonates in N → O transition. Interestingly, the D196N mutation increases 15-syn-retinal at the expense of 15-anti, which is the predominant isomer in the wild type, and abolishes the peak current in electrophysiological measurements. This suggests that the peak current is formed by 15-anti species, whereas 15-syn species contribute only to the stationary current.

Frequency Dependent Non- Thermal Effects of Oscillating Electric Fields in the Microwave Region on the Properties of a Solvated Lysozyme System: A Molecular Dynamics Study.

The use of microwaves in every day's applications raises issues regarding the non thermal biological effects of microwaves. In this work we employ molecular dynamics simulations to advance further the dielectric studies of protein solutions in the case of lysozyme, taking into consideration possible frequency dependent changes in the structural and dynamic properties of the system upon application of electric field in the microwave region. The obtained dielectric spectra are identical with those derived in our previous work using the Fröhlich-Kirkwood approach in the framework of the linear response theory. Noticeable structural changes in the protein have been observed only at frequencies near its absorption maximum. Concerning Cα position fluctuations, different frequencies affected different regions of the protein sequence. Furthermore, the influence of the field on the kinetics of protein-water as well as on the water-water hydrogen bonds in the first hydration shell has been studied; an extension of the Luzar-Chandler kinetic model was deemed necessary for a better fit of the applied field results and for the estimation of more accurate hydrogen bond lifetime values.

Amide-directed photoredox-catalysed C-C bond formation at unactivated sp3 C-H bonds.

Carbon-carbon (C-C) bond formation is paramount in the synthesis of biologically relevant molecules, modern synthetic materials and commodity chemicals such as fuels and lubricants. Traditionally, the presence of a functional group is required at the site of C-C bond formation. Strategies that allow C-C bond formation at inert carbon-hydrogen (C-H) bonds enable access to molecules that would otherwise be inaccessible and the development of more efficient syntheses of complex molecules. Here we report a method for the formation of C-C bonds by directed cleavage of traditionally non-reactive C-H bonds and their subsequent coupling with readily available alkenes. Our methodology allows for amide-directed selective C-C bond formation at unactivated sp3 C-H bonds in molecules that contain many such bonds that are seemingly indistinguishable. Selectivity arises through a relayed photoredox-catalysed oxidation of a nitrogen-hydrogen bond. We anticipate that our findings will serve as a starting point for functionalization at inert C-H bonds through a strategy involving hydrogen-atom transfer.

Competition between hydrogen bonding and protein aggregation in neuronal-like cells under exposure to 50 Hz magnetic field.

PURPOSE: To investigate the role of hydrogen bonding and protein unfolding in human SH-SY5Y neuronal-like cells under exposure to a 50 Hz magnetic field (MF) at the intensity of 1 mT. MATERIALS AND METHODS: Neuronal-like cells were placed into an incubator in a 5% CO2/95% air humidified at the temperature of 37.1 °C and exposed for 4 h to a 50 Hz MF at 1 mT. The exposure system consisted of two Helmholtz coils driven by AC voltage at 50 Hz. Exposed and control samples were studied using Fourier Transform Infrared (FTIR) Spectroscopy. RESULTS: The vibration bands of the methylene group increased significantly after 4 h of exposure. A significant shift to low energies of the Amide I band and an increase in the intensity of the parallel and antiparallel ß-sheet structures with respect to the α-helix component were observed after exposure. The Amide II frequency did not change significantly whereas a relative increase of its integrated area with respect to Amide I mode occurred after exposure. CONCLUSIONS: These results can be explained assuming that both the mechanisms of protein aggregation as well as the increase in hydrogen bonding occurred in neuronal-like cells under exposure to a 50 Hz MF.

[On a Possible Mechanism of the Effect of Microwave Radiation on Biological Macromolecules].

A model describing the process of dissociation of hydrogen bonding in water clusters when irradiated by electromagnetic field in the microwave range is suggested. The model is also applicable for the case of rupture of the covalent bond of the water molecule cluster. If the energy absorption occurs at the interface of water and polymer clusters (e.g., DNA, chitosan), degradation of the polymer chain is possible.

The linker between LOV2-Jα and STK plays an essential role in the kinase activation by blue light in Arabidopsis phototropin1, a plant blue light receptor.

Phototropin (phot), a blue light receptor in plants, is composed of several domains: LOV1, LOV2, and a serine/threonine kinase (STK). LOV2 is the main regulator of light activation of STK. However, the detailed mechanism remains unclear. In this report, we focused on the linker region between LOV2 and STK excluding the Jα-helix. Spectroscopy and a kinase assay for the substituents in the linker region of Arabidopsis phot1 LOV2-STK indicated that the linker is involved in the activation of STK. A putative module in the middle of the linker would be critical for intramolecular signaling and/or regulation of STK.

Reconstruction of Block Copolymer Micelles to Long-Range Ordered Dense Nanopatterns Via Light-Tunable Hydrogen-Bonding Association.

Controlling the orientation and long-range order of nanostructures is a key issue in the self-assembly of block copolymer micelles. Herein, a versatile strategy is presented to transform one-component oxime-based block copolymer micelles into long-range ordered dense nanopatterns. Photoisomerization provides a straightforward and versatile approach to convert the hydrogen-bonding association from inward dimerization (E-type oxime motifs, slightly desolvated in ethyl acetate) into outward interchain association (Z-type ones, highly desolvated in ethyl acetate). This increases the glass transition temperature in bulk and converts swollen micelles into compact spherical micelles in solution. The reconstruction of these micelles on various substrates demonstrates that the phase transformation enables reconstruction of spherical micelles into mesoscopic sheets, nanorods, nanoworms, nanowires, networks, and eventually into long-range ordered and densely packed textile-like and lamellar nanopatterns on a macroscopic scale by adjusting E/Z-oxime ratio and solvent-evaporation rate.

Photo-physical properties of 2-(1-ethynylpyrene)-adenosine: influence of hydrogen bonding on excited state properties.

The photo-physical properties of 2-(1-ethynylpyrene)-adenosine (PyA), a fluorescent probe for RNA dynamics, were examined by solvation studies. The excited-state dynamics display the influence of the vicinity on the spectral features. Combining improved transient absorption and streak camera measurements along with a new analysis method provide a detailed molecular picture of the photophysics. After intramolecular vibrational energy redistribution (IVR), two distinct states are observed. Solvent class (protic/aprotic) and permittivity strongly affect the properties of these states and their population ratio. As a result their emission spectrum is altered, while the fluorescence quantum yield and the overall lifetime remain nearly unchanged. Consequently, the hitherto existing model of the photophysics is herein refined and extended. The findings can serve as basis for improving the information content of measurements with PyA as a label in RNA.

[Ultrasonic cleavage of DNA in complexes with Ag(I), Cu(II), Hg(II)].

We investigated a phenomenon of ultrasonic cleavage of DNA complexed with transition metal cations Ag(I), Cu(II) and Hg(II). We found the statistically significant dependence of relative intensity of cleavage on cation type and concentration. Each cation may cause two different types of distortion in the DNA double-helix depending on whether it binds to major or minor DNA groove. The intensity of ultrasonic cleavage decreases if cation binds to the major DNA groove; the intensity of cleavage increases if cation binds to the minor DNA groove and disturbs the hydrogen bonds of complementary base pairs or it intercalates between bases. Both types of DNA distortion can affect the intensity of N-S interconversion of deoxyribose.

Crystal structures of thiobarbiturate with N-phenylalkyl group and its photocyclization product.

The photo-irradiation of thiobarbiturates (1) with an N-phenylalkyl group gives bicyclic fused pyrimidine derivatives (2) through a Norrish type II reaction. The crystal structures of dihydro-1,5,5-trimethyl-3-(3-phenylpropyl)-2-thioxo-4,6(1H,5H)-pyrimidinedione (1a) and the photocyclization product (2b) of dihydro-1,5,5-trimethyl-3-(3-phenylbutyl)-2-thioxo-4,6(1H,5H)-pyrimidinedione (1b) were elucidated by X-ray crystallographic analysis. The photocyclization product (2b) was determined to be 1,6,7,8-tetrahydro-1,3,3-trimethyl-9-phenyl-2H-pyrido[1,2-a]pyrimidine-2,4(3H)-dione.

Anion responsive dibenzoyl-L-cystine and luminescent lanthanide soft material.

A mild sol-gel technique was used to incorporate terbium dibenzoyl-L-cystine complex into silica and green luminescent hybrid material was fabricated. (1)H NMR and fluorescence spectroscopy revealed the hybrids can recognize F(-) anions through hydrogen bonding formation and had no sense in binding other halide or HSO(4)(-). Furthermore, a luminescent hydrogel was successfully designed by immobilizing a terbium activated phosphor (Gd(0.1)Ce(0.9)PO(4):Tb) into molecular hydrogelator (dibenzoyl-L-cystine). The Tb(III) emission in hydrogel media gave a distinguished enhancement based on temperature increase and the function conforms to exponential equation y = 1160.6 exp(0.03x). The stability of the green luminescent gel was rather excellent and the reversibility of the gel can be recycled at least five times.

Hydrogen bond perturbation in hen egg white lysozyme by external electromagnetic fields: a nonequilibrium molecular dynamics study.

Nonequilibrium molecular dynamics simulations of a charge-neutral mutant of hen egg white lysozyme have been performed at 300 K and 1 bar in the presence of external microwave fields (2.45 to 100 GHz) of an rms electric field intensity of 0.05 V Å(-1). A systematic study was carried out of the distributions of persistence times and energies of each intraprotein hydrogen bond in between breakage and reformation, in addition to overall persistence over 20 ns simulations, vis-á-vis equilibrium, zero-field conditions. It was found that localized translational motion for formally charged residues led to greater disruption of associated hydrogen bonds, although induced rotational motion of strongly dipolar residues also led to a degree of hydrogen bond perturbation. These effects were most apparent in the solvent exposed exterior of hen egg white lysozyme, in which the intraprotein hydrogen bonds tend to be weaker.

On the hydration of the phosphocholine headgroup in aqueous solution.

The hydration of the phosphocholine headgroup in 1,2-dipropionyl-sn-glycero-3-phosphocholine (C(3)-PC) in solution has been determined by using neutron diffraction enhanced with isotopic substitution in combination with computer simulation techniques. The atomic scale hydration structure around this head group shows that both the -N(CH(3))(3) and -CH(2) portions of the choline headgroup are strongly associated with water, through a unique hydrogen bonding regime, where specifically a hydrogen bond from the C-H group to water and a strong association between the water oxygen and N(+) atom in solution have both been observed. In addition, both PO(4) oxygens (P=O) and C=O oxygens are oversaturated when compared to bulk water in that the average number of hydrogen bonds from water to both X=O oxygens is about 2.5 for each group. That water binds strongly to the glycerol groups and is suggestive that water may bind to these groups when phosophotidylcholine is embedded in a membrane bilayer.

Ultrasound stimulus effect on hydrogen bonding in networked alumina and polyacrylic acid slurry.

The influence of ultrasound (US) on the viscosity of aqueous slurry composing polyacrylic acid (PAA) and alumina was studied. For exposure to an aqueous slurry solution, US waves emitted at three different frequencies of 28, 45, and 100 kHz were used. Results show that the stimulus effect of US on viscosity change was a breakage of the hydrogen bonding networks of alumina and PAA in the slurry solution. That decrease in viscosity was enhanced strongly by US exposure as the output power was increased from 175 to 300 W. In addition, a lower US frequency was effective for slurry viscosity reduction. The reduced viscosity of the slurry also depended on the solution pH. After US was stopped, the viscosity increased gradually and recovered to its original value within about 15 min. The stimulus effect on the viscosity change was cycled by US.

Role of the carbonyl group of the (6-4) photoproduct in the (6-4) photolyase reaction.

The (6-4) photoproduct, which is one of the major UV-induced DNA lesions, causes carcinogenesis with high frequency. The (6-4) photolyase is a flavoprotein that can restore this lesion to the original bases, but its repair mechanism has not been elucidated. In this study, we focused on the interaction between the enzyme and the 3' pyrimidone component of the (6-4) photoproduct and prepared a substrate analogue in which the carbonyl group, a hydrogen-bond acceptor, was replaced with an imine, a hydrogen-bond donor, to investigate the involvement of this carbonyl group in the (6-4) photolyase reaction. UV irradiation of oligodeoxyribonucleotides containing a single thymine-5-methylisocytosine site yielded products with absorption bands at wavelengths longer than 300 nm, similar to those obtained from the conversion of the TT site to the (6-4) photoproduct. Nuclease digestion, MALDI-TOF mass spectrometry, and the instability of the products indicated the formation of the 2-iminopyrimidine-type photoproduct. Analyses of the reaction and the binding of the (6-4) photolyase using these oligonucleotides revealed that this imine analogue of the (6-4) photoproduct was not repaired by the (6-4) photolyase, although the enzyme bound to the oligonucleotide with considerable affinity. These results indicate that the carbonyl group of the 3' pyrimidone ring plays an important role in the (6-4) photolyase reaction. On the basis of these results, we discuss the repair mechanism.