Dynamics of charge transfer along an oligonucleotide at finite temperature

The quantum-statistical approach was used to describe the charge transfer in nucleotide sequences. The results of numerical modeling for hole transfer in the GTTGGG sequence with background temperature noise are given. It was shown that, since guanine has an oxidation potential lower than thymine, the hole created at the G donor in this sequence passes through the thymine barrier into the guanine triplet (acceptor) at a time of approximately 10 ps at a temperature of 37 degrees C.     

Charge Transport in Pure and Mixed Phases in Organic Solar Cells

In organic solar cells continuous donor and acceptor networks are considered necessary for charge extraction, whereas discontinuous neat phases and molecularly mixed donor–acceptor phases are generally regarded as detrimental. However, the impact of different levels of domain continuity, purity, and donor–acceptor mixing on charge transport remains only semiquantitatively described. Here, cosublimed donor–acceptor mixtures, where the distance between the donor sites is varied in a controlled manner from homogeneously diluted donor sites to a continuous donor network are studied. Using transient measurements, spanning from sub‐picoseconds to microseconds photogenerated charge motion is measured in complete photovoltaic devices, to show that even highly diluted donor sites (5.7%–10% molar) in a buckminsterfullerene matrix enable hole transport. Hopping between isolated donor sites can occur by long‐range hole tunneling through several buckminsterfullerene molecules, over distances of up to ≈4 nm. Hence, these results question the relevance of “pristine” phases and whether a continuous interpenetrating donor–acceptor network is the ideal morphology for charge transport.     

Luminescence quenching of Ru-labeled oligonucleotides by targeted complementary strands.

The yield of hole injection into guanines of different oligonucleotide duplexes by a photooxidizing tethered Ru(II) complex is examined by measuring the luminescence quenching of the excited complex. This yield is investigated as a function of the anchoring site of the complex (on a thymine nucleobase in the middle of the sequence or on the 5' terminal phosphate) and the number and position of the guanine bases as compared with the site of attachment of the Ru(II) compound. In contrast to other studies, the tethered complex, [Ru(tap)(2)(dip)](2+), is a non-intercalating compound and has been shown previously to produce an irreversible photocrosslinking between the two strands as the ultimate step of hole injection. The study of luminescence quenching of the anchored complex by emission intensity and lifetime measurements for the different duplexes indicates that a direct contact between the complex and the guanine nucleobase is needed for the electron transfer to take place. Moreover, for none of the sequences a clear contribution of a static quenching is evidenced independently of the two types of attachment of the [Ru(tap)(2)(dip)](2+) complex to the oligonucleotide. A comparison of the fastest hole-injection process by electron transfer to the excited anchored [Ru(tap)(2)(dip)](2+), with the rate of the photo-electron transfer between the same complex free in solution and guanosine-5'-monophosphate, indicates that the hole injection by the anchored complex is slower by a factor of 10 at least. A bad overlap between donor and acceptor orbitals is probably the cause of this slow rate, which could be attributed to some steric hindrance induced by the complex linker.     

Functional cloning, heterologous expression, and purification of two different N-deoxyribosyltransferases from Lactobacillus helveticus

Lactobacillus helveticus contains two types of N-deoxyribosyltransferases: DRTase I catalyzes the transfer of 2'-deoxyribose between purine bases exclusively whereas DRTase II is able to transfer the 2'-deoxyribose between two pyrimidine or between pyrimidine and purine bases. An Escherichia coli strain, auxotrophic for guanine and unable to use deoxyguanosine as source of guanine, was constructed to clone the corresponding genes. By screening a genomic bank for the production of guanine, the L. helveticus ptd and ntd genes coding for DRTase I and II, respectively, were isolated. Although the two genes have no sequence similarity, the two deduced polypeptides display 25.6% identity, with most of the residues involved in substrate binding and the active site nucleophile Glu-98 being conserved. Overexpression and purification of the two proteins shows that DRTase I is specific for purines with a preference for deoxyinosine (dI) > deoxyadenosine > deoxyguanosine as donor substrates whereas DRTase II has a strong preference for pyrimidines as donor substrates and purines as base acceptors. Purine analogues were substrates as acceptor bases for both enzymes. Comparison of DRTase I and DRTase II activities with dI as donor or hypoxanthine as acceptor and colocalization of the ptd and add genes suggest a specific role for DRTase I in the metabolism of dI.     

Enzymatic synthesis of 2'-deoxyguanosine with nucleoside deoxyribosyltransferase-II

Nucleoside deoxyribosyltransferase-II (NdRT-II) of Lactobacillus helveticus, which catalyzes the transfer of a glycosyl residue from a donor deoxyribonucleoside to an acceptor base, has a broad specificity for the acceptor bases. Six-substituted purines were found to be substrates as acceptor bases for NdRT-II. Using this property of the enzyme, we established a practical procedure for enzymatic synthesis of 2'-deoxyguanosine (dGuo), consisting of the transglycosylation from thymidine to 6-substituted purine (2-amino-6-chloropurine; ACP) instead of natural guanine and the conversion of 2-amino-6-chloropurine-2'-deoxyriboside (ACPdR) to dGuo with bacterial adenosine deaminase. Through the successive reactions, dGuo was synthesized in high yield.     

Glucosyl transferase activity of bovine galactosyl transferase.

Bovine galactosyl transferase was found to utilize UDPglucose as a substrate and elicit disaccharide biosynthesis with glucose and N-acetylglucosamine as acceptors. The relative rate of glucosyl transferase with N-acetylglucosamine as acceptor was 0.3%, the rate for N-acetyllactosamine biosynthesis. This activity was also evidenced indirectly from NMR water proton relaxation experiments, and from Mn(II) ESR experiments. In direct experiments with radioactive UDPglucose, paper chromatography showed a product which migrated with cellobiose when glucose was the acceptor and a new, glucose-containing product which resulted when GlcNAc was the acceptor. Despite this marginally expanded specificity of the donor site, spin-label experiments with a covalently bound UDPgalactose analog reaffirmed the restrictive nature of the donor site against this non-glycosyl-like analog.     

Donor-acceptor model of electron transfer through proteins

It is shown that the interaction of proteins with a polar solvent leads to the formation of electron donor groups in the proteins. The highest occupied level of these groups in situated near the bottom of the conduction energy band of protein. The transfer of an electron from the donor group through the conduction band of protein to the spatially removed acceptor group is considered including the possible relaxation processes.     

Lateral diffusion coefficients in membranes measured by resonance energy transfer and a new algorithm for diffusion in two dimensions

We describe measurements of lateral diffusion in membranes using resonance energy transfer. The donor was a rhenium (Re) metal-ligand complex lipid, which displays a donor decay time near 3 micros. The long donor lifetime resulted in an ability to measure lateral diffusion coefficient below 10(-8) cm(2)/s. The donor decay data were analyzed using a new numerical algorithm for calculation of resonance energy transfer for donors and acceptors randomly distributed in two dimensions. An analytical solution to the diffusion equation in two dimensions is not known, so the equation was solved by the relaxation method in Laplace space. This algorithm allows the donor decay in the absence of energy transfer to be multiexponential. The simulations show that mutual lateral diffusion coefficients of the donor and acceptor on the order of 10(-8) cm(2)/s are readily recovered from the frequency-domain data with donor decay times on the microsecond timescale. Importantly, the lateral diffusion coefficients and acceptor concentrations can be recovered independently despite correlation between these parameters. This algorithm was tested and verified using the donor decays of a long lifetime rhenium lipid donor and a Texas red-lipid acceptor. Lateral diffusion coefficients ranged from 4.4 x 10(-9) cm(2)/s in 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG) at 10 degrees C to 1.7 x 10(-7) cm(2)/s in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at 35 degrees C. These results demonstrated the possibility of direct measurements of lateral diffusion coefficients using microsecond decay time luminophores.     

Intermembrane phospholipid transfer mediated by cell-free extracts of Rhodopseudomonas sphaeroides.

The transfer of phospholipids between two membrane substrates catalyzed by a soluble protein fraction from Rhodopseudomonas sphaeroides has been demonstrated. The assay employs purified intracytoplasmic membrane (ICM) vesicles derived from cells of R. sphaeroides grown on [3H]acetate as the phospholipid donor substrate and phosphatidylcholine (70%)/phosphatidylethanolamine (30%) unilamellar liposomes containing [14C]triolein, a nontransferable marker, as the acceptor substrate for transferred phospholipids. Incubation of these two membrane substrates with a 40 to 80% (NH4)2SO4 protein fraction from R. sphaeroides results in the transfer of tritium-labeled ICM phospholipids to the acceptor membrane substrate. Upon completion of the incubation period, the donor ICM vesicles are quantitatively separated from the acceptor liposomes by precipitation with antibody prepared against whole, purified ICM vesicles. Phospholipid transfer is linear with respect to time and protein concentration, is inhibited by trypsin and heat, and shows an absolute dependence upon the presence of acceptor liposomes and the 40 to 80% (NH4)2SO4 protein fraction. Control experiments indicate that no fusion of the donor and acceptor membrane occurs during the incubation period and that, following prolonged incubation there is no detectable degradation of the labeled lipid components. Preliminary data on the phospholipid specificity of the transfer reaction is also presented.     

Distance-dependent photoinduced electron transfer in synthetic single-strand and hairpin DNA

 The singlet state of stilbene-4,4′-dicarboxamide can serve as a fluorescent probe of both DNA conformation and electron transfer. Covalent incorporation of the stilbene-dicarboxamide into DNA structures with restricted conformational mobility results in inhibition of stilbene isomerization and an increase in its fluorescence quantum yield and lifetime. The fluorescence of stilbenedicarboxamide is selectively quenched by proximate guanine, but not by the three other DNA nucleobases. Selective quenching occurs via an electron transfer mechanism in which stilbene serves as the electron acceptor and guanine as the electron donor. Kinetic analysis of the distance dependence of electron transfer in stilbene-bridged hairpins suggests that duplex DNA is more effective than proteins as a medium for electron transfer, but that it does not function as a molecular wire. Received, accepted: 5 January 1998     

Glucosyl transferase activity of bovine galactosyl transferase

Bovine galactosyl transferase was found to utilize UDPglucose as a substrate and elicit disaccharide biosynthesis with glucose and N-acetylglucosamine as acceptors. The relative rate of glycosyl transferase with N-acetylglucosamine as acceptor was 0.3%, the rate for N-acetyllactosamine biosynthesis. This activity was also evidenced indirectly from NMR water proton relaxation experiments, and from Mn(II) ESR experiments. In direct experiments with radioactive UDPglucose, paper chromatography showed a product which migrated with cellobiose when glucose was the acceptor and a new, glucose-containing product which resulted when GlcNAc was the acceptor.Despite this marginally expanded specificity of the donor site, spin-label experiments with a covalently bound UDPgalactose analog reaffirmed the restrictive nature of the donor site against this non-glycosyl-like analog.     

The recovery of the polymerizability of Lys-61-labelled actin by the addition of phalloidin. Fluorescence polarization and resonance-energy-transfer measurements

Modification of Lys-61 in actin with fluorescein-5-isothiocyanate (FITC) blocks actin polymerization [Burtnick, L. D. (1984) Biochim. Biophys. Acta 791, 57-62]. FITC-labelled actin recovered its ability to polymerize on addition of phalloidin. The polymers had the same characteristic helical thread-like structure as normal F-actin and the addition of myosin subfragment-1 to the polymers formed the characteristic arrowhead structure in electron microscopy. The polymers activated the ATPase activity of myosin subfragment-1 as efficiently as normal F-actin. These results indicate that Lys-61 is not directly involved in an actin-actin binding region nor in myosin binding site. From static fluorescence polarization measurements, the rotational relaxation time of FITC-labelled actin filaments was calculated to be 20 ns as the value reduced in water at 20 degrees C, while any rotational relaxation time of 1,5-IAEDANS bound to Cys-374 on F-actin in the presence of a twofold molar excess of phalloidin could not be detected by static polarization measurements under the same conditions. This indicates that the Lys-61 side chain is extremely mobile even in the filamentous structure. Fluorescence resonance energy transfer between the donor 1,5-IAEDANS bound to SH1 of myosin subfragment-1 and the acceptor fluorescein-5-isothiocyanate bound to Lys-61 of actin in the rigor complex was measured. The transfer efficiency was 0.39 +/- 0.05 which corresponds to the distance of 5.2 +/- 0.1 nm, assuming that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime and that the transfer occurs between a single donor and an acceptor.     

Determining the optimum model parameters for oligosaccharide production efficiency using response surface integrated particle swarm optimization method: an experimental validation study

Abstract

Glucansucrases (GTFs) catalyzes the synthesis of α-glucans from sucrose and oligosaccharides in the presence of an acceptor sugar by transferring glucosyl units to the acceptor molecule with different linkages. The acceptor reactions can be affected by several parameters and this study aimed to determine the optimal reaction parameters for the production of glucansucrase-based oligosaccharides using sucrose and maltose as the donor and acceptor sugars, respectively via a hybrid technique of Response Surface Method (RSM) and Particle Swarm Optimization (PSO). The experimental design was performed using Central Composite Design and the tested parameters were enzyme concentration, acceptor:donor ratio and the reaction period. The optimization studies showed that enzyme concentration was the most effective parameter for the final oligosaccharides yields. The optimal values of the significant parameters determined for enzyme concentration and acceptor:donor ratio were 3.45?U and 0.62, respectively. Even the response surface plots for input parameters verified the PSO results, an experimental validation study was performed for the reverification. The experimental verification results obtained were also consistent with the PSO results. These findings will help our understanding in the role of different parameters for the production of oligosaccharides in the acceptor reactions of GTFs.     

Local Intermolecular Order Controls Photoinduced Charge Separation at Donor/Acceptor Interfaces in Organic Semiconductors

How free charge is generated at organic donor–acceptor interfaces is an important question, as the binding energy of the lowest energy (localized) charge transfer states should be too high for the electron and hole to escape each other. Recently, it has been proposed that delocalization of the electronic states participating in charge transfer is crucial, and aggregated or otherwise locally ordered structures of the donor or the acceptor are the precondition for this electronic characteristic. The effect of intermolecular aggregation of both the polymer donor and fullerene acceptor on charge separation is studied. In the first case, the dilute electron acceptor triethylsilylhydroxy‐1,4,8,11,15,18,22,25‐octabutoxyphthalocyaninatosilicon(IV) (SiPc) is used to eliminate the influence of acceptor aggregation, and control polymer order through side‐chain regioregularity, comparing charge generation in 96% regioregular (RR‐) poly(3‐hexylthiophene) (P3HT) with its regiorandom (RRa‐) counterpart. In the second case, ordered phases in the polymer are eliminated by using RRa‐P3HT, and phenyl‐C₆₁‐butyric acid methyl ester (PC₆₁BM) is used as the acceptor, varying its concentration to control aggregation. Time‐resolved microwave conductivity, time‐resolved photoluminescence, and transient absorption spectroscopy measurements show that while ultrafast charge transfer occurs in all samples, long‐lived charge carriers are only produced in films with intermolecular aggregates of either RR‐P3HT or PC₆₁BM, and that polymer aggregates are just as effective in this regard as those of fullerenes.     

Light-adaptation of photosystem II is mediated by the plastoquinone pool

During the first few enzymatic turnovers after dark-adaptation of photosystem II (PSII), the relaxation rate of the EPR signals from the Mn cluster and Y(D)(*) are significantly enhanced. This light-adaptation process has been suggested to involve the appearance of a new paramagnet on the PSII donor side [Peterson, S., Ahrling, K., H?gblom, J., and Styring, S. (2003) Biochemistry 42, 2748-2758]. In the present study, a correlation is established between the observed relaxation enhancement and the redox state of the quinone pool. It is shown that the addition of quinol to dark-adapted PSII membrane fragments induces relaxation enhancement already after a single oxidation of the Mn, comparable to that observed after five oxidations in samples with quinones (PPBQ or DQ) added. The saturation behavior of Y(D)(*) revealed that with quinol added in the dark, a single flash was necessary for the relaxation enhancement to occur. The quinol-induced relaxation enhancement of PSII was also activated by illumination at 200 K. Whole thylakoids, with no artificial electron acceptor present but with an intact plastoquinone pool, displayed the same relaxation enhancement on the fifth flash as membrane fragments with exogenous quinones present. We conclude that (i) reduction of the quinone pool induces the relaxation enhancement of the PSII donor-side paramagnets, (ii) light is required for the quinol to effect the relaxation enhancement, and (iii) light-adaptation occurs in the intact thylakoid system, when the endogenous plastoquinone pool is gradually reduced by PSII turnover. It seems clear that a species on the PSII donor side is reduced by the quinol, to become a potent paramagnetic relaxer. On the basis of XANES reports, we suggest that this species may be the Mn ions not involved in the cyclic redox changes of the oxygen-evolving complex.     

On the possibility of long-wavelength long-lifetime high-quantum-yield luminophores

We describe an approach to creating a new class of luminophores which display both long wavelength emissions exceeding 600 nm and long lifetimes. These luminophores are based on resonance energy transfer (RET) from a long lifetime donor to a short lifetime but long wavelength acceptor. We demonstrated the possibility of obtaining these desirable spectral properties using donors and acceptors noncovalently bound to DNA. The donor was a ruthenium (Ru) metal-ligand complex in which one of the diimine ligands intercalated into double-helix DNA. The acceptors were either nile blue, TOTO-3, or TO-PRO-3. Upon binding of the acceptor to donor-labeled DNA, we found that the acceptor quantum yield was remarkably enhanced so that the wavelength-integrated intensities of the donor and acceptor bound to DNA were many-fold greater than the intensity of the donor and acceptor alone when separately bound to DNA. The origin of this effect is efficient energy transfer from the donor. Under these conditions the effective overall quantum yield approaches that of the acceptor. Importantly, the increased quantum yield can be obtained while maintaining usefully long apparent acceptor lifetimes of 30 to 80 ns. The effect of an increased quantum yield from a low quantum yield donor may find use in assays to detect macromolecular binding interactions. These results suggest the synthesis of covalently linked donor-acceptor pairs with the desirable spectral properties of long wavelength emission, high quantum yield, and moderately long lifetimes for gated detection.     

Fluorescence energy transfer between Ca2+ transport ATPase molecules in artificial membranes.

The purified ATPase of sarcoplasmic reticulum was covalently labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) or with iodoacetamidofluorescein (IAF). In reconstituted vesicles containing both types of ATPase molecules fluorescence energy transfer was observed from the IAEDANS (donor) to the IAF (acceptor) fluorophore as determined by the ratio of donor and acceptor fluorescence intensities, and by nanosecond decay measurements of donor fluorescence in the presence or absence of the acceptor. The observed energy transfer may arise by random collisions between ATPase molecules due to Brownian motion or by formation of complexes containing several ATPase molecules. Experimental distinction between these two models of energy transfer is possible based on predictions derived from mathematical models. Up to tenfold dilution of the lipid phase of reconstituted vesicles with egg lecithin had no measurable effect upon the energy transfer, suggesting that random collision between ATPase molecules in the lipid phase is not the principal cause of the observed effect. Addition of unlabeled ATPase in five- to tenfold molar excess over the labeled molecules abolished energy transfer. These observations together with electron microscopic and chemical cross-linking studies support the existence of ATPase oligomers in the membrane with sufficiently long lifetimes for energy transfer to occur. A hypothetical equilibrium between monomeric and tetrameric forms of the ATPase governed by the membrane potential is proposed as the structural basis of the regulation of Ca uptake and release by sarcoplasmic reticulum membranes during muscle contraction and relaxation.     

Characterization of the primary radical pair in reaction centers of Heliobacillus mobilis by 13C photo-CIDNP MAS NMR

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in membrane fragments of heliobacterium Heliobacillus mobilis without further isolation by (13)C magic-angle spinning (MAS) solid-state NMR under continuous illumination with white light. In the (13)C photo-CIDNP MAS NMR spectra of heliobacterial membrane fragments, two sets of signals are observed, allowing characterization of the primary radical pair. One set, showing enhanced absorptive (positive) signals, arises from the BChl g donor, while the set of emissive (negative) signals is assigned to the 8(1)-hydroxy Chl a acceptor. Hence, under these sample conditions, both donor and acceptor sides are either monomeric or composed of identical cofactors. The occurrence of the differential relaxation (DR) mechanism suggests a donor triplet lifetime in the microsecond range. It appears that the occurrence of the solid-state photo-CIDNP effect is a general feature of primary radical pairs in natural photosynthesis.