Distribution Of Donor-acceptor Distances Research Articles

Hydride Transfer Mechanism of Enzymatic Sugar Nucleotide C2 Epimerization Probed with a Loose-Fit CDP-Glucose Substrate.

Transient oxidation–reduction through hydride transfer with tightly bound NAD coenzyme is used by a large class of sugar nucleotide epimerases to promote configurational inversion of carbon stereocenters in carbohydrate substrates. A requirement for the epimerases to coordinate hydride abstraction and re-addition with substrate rotation in the binding pocket poses a challenge for dynamical protein conformational selection linked to enzyme catalysis. Here, we studied the thermophilic C2 epimerase from Thermodesulfatator atlanticus (TaCPa2E) in combination with a slow CDP-glucose substrate (kcat ≈ 1.0 min–1; 60 °C) to explore the sensitivity of the enzymatic hydride transfer toward environmental fluctuations affected by temperature (20–80 °C). We determined noncompetitive primary kinetic isotope effects (KIE) due to 2H at the glucose C2 and showed that a normal KIE on the kcat (Dkcat) reflects isotope sensitivity of the hydrogen abstraction to enzyme-NAD+ in a rate-limiting transient oxidation. The Dkcat peaked at 40 °C was 6.1 and decreased to 2.1 at low (20 °C) and 3.3 at high temperature (80 °C). The temperature profiles for kcat with the 1H and 2H substrate showed a decrease in the rate below a dynamically important breakpoint (∼40 °C), suggesting an equilibrium shift to an impaired conformational landscape relevant for catalysis in the low-temperature region. Full Marcus-like model fits of the rate and KIE profiles provided evidence for a high-temperature reaction via low-frequency conformational sampling associated with a broad distribution of hydride donor–acceptor distances (long-distance population centered at 3.31 ± 0.02 Å), only poorly suitable for quantum mechanical tunneling. Collectively, dynamical characteristics of TaCPa2E-catalyzed hydride transfer during transient oxidation of CDP-glucose reveal important analogies to mechanistically simpler enzymes such as alcohol dehydrogenase and dihydrofolate reductase. A loose-fit substrate (in TaCPa2E) resembles structural variants of these enzymes by extensive dynamical sampling to balance conformational flexibility and catalytic efficiency.

Evolution of Optimized Hydride Transfer Reaction and Overall Enzyme Turnover in Human Dihydrofolate Reductase.

Evolution of dihydrofolate reductase (DHFR) has been studied using the enzyme from Escherichia coli DHFR (ecDHFR) as a model, but less studies have used the enzyme from Homo sapiens DHFR (hsDHFR). Each enzyme maintains a short and narrow distribution of hydride donor-acceptor distances (DAD) at the tunneling ready state (TRS). Evolution of the enzyme was previously studied in ecDHFR where three key sites were identified as important to the catalyzed reaction. The corresponding sites in hsDHFR are F28, 62-PEKN, and 26-PPLR. Each of these sites was studied here through the creation of mutant variants of the enzyme and measurements of the temperature dependence of the intrinsic kinetic isotope effects (KIEs) on the reaction. F28 is mutated first to M (F28M) and then to the L of the bacterial enzyme (F28L). The KIEs of the F28M variant are larger and more temperature-dependent than wild-type (WT), suggesting a broader and longer average DAD at the TRS. To more fully mimic ecDHFR, we also study a triple mutant of the human enzyme (F32L-PP26N-PEKN62G). Remarkably, the intrinsic KIEs, while larger in magnitude, are temperature-independent like the WT enzymes. We also construct deletion mutations of hsDHFR removing both the 62-PEKN and 26-PPLR sequences. The results mirror those described previously for insertion mutants of ecDHFR. Taken together, these results suggest a balancing act during DHFR evolution between achieving an optimal TRS for hydride transfer and preventing product inhibition arising from the different intercellular pools of NADPH and NADP+ in prokaryotic and eukaryotic cells.

Differential flexibility leading to crucial microelastic properties of asymmetric lipid vesicles for cellular transfection: A combined spectroscopic and atomic force microscopy studies

The role of microscopic elasticity of nano-carriers in cellular uptake is an important aspect in biomedical research. Herein we have used AFM nano-indentation force spectroscopy and Förster resonance energy transfer (FRET) measurements to probe microelastic properties of three novel cationic liposomes based on di-alkyl dihydroxy ethyl ammonium chloride based lipids having asymmetry in their hydrophobic chains (Lip1818, Lip1814 and Lip1810). AFM data reveals that symmetry in hydrophobic chains of a cationic lipid (Lip1818) imparts higher rigidity to the resulting liposomes than those based on asymmetric lipids (Lip1814 and Lip1810). The stiffness of the cationic liposomes is found to decrease with increasing asymmetry in the hydrophobic lipid chains in the order of Lip1818 > Lip1814 > lip1810. FRET measurements between Coumarin 500 (Donor) and Merocyanine 540 (Acceptor) have revealed that full width at half-maxima (hw) of the probability distribution (P(r)) of donor-acceptor distance (r), increases in an order Lip1818 < Lip1814 < Lip1810 with increasing asymmetry of the hydrophobic lipid chains. This increase in width (hw) of the donor-acceptor distance distributions is reflective of increasing flexibility of the liposomes with increasing asymmetry of their constituent lipids. Thus, the results from AFM and FRET studies are complementary to each other and indicates that an increase in asymmetry of the hydrophobic lipid chains increases elasticity and or flexibility of the corresponding liposomes. Cell biology experiments confirm that liposomal flexibility or rigidity directly influences their cellular transfection efficiency, where Lip1814 is found to be superior than the other two liposomes manifesting that a critical balance between flexibility and rigidity of the cationic liposomes is key to efficient cellular uptake. Taken together, our studies reveal how asymmetry in the molecular architecture of the hydrophobic lipid chains influences the microelastic properties of the liposomes, and hence, their cellular uptake efficiency.

Inferring properties of disordered chains from FRET transfer efficiencies.

Förster resonance energy transfer (FRET) is a powerful tool for elucidating both structural and dynamic properties of unfolded or disordered biomolecules, especially in single-molecule experiments. However, the key observables, namely, the mean transfer efficiency and fluorescence lifetimes of the donor and acceptor chromophores, are averaged over a broad distribution of donor-acceptor distances. The inferred average properties of the ensemble therefore depend on the form of the model distribution chosen to describe the distance, as has been widely recognized. In addition, while the distribution for one type of polymer model may be appropriate for a chain under a given set of physico-chemical conditions, it may not be suitable for the same chain in a different environment so that even an apparently consistent application of the same model over all conditions may distort the apparent changes in chain dimensions with variation of temperature or solution composition. Here, we present an alternative and straightforward approach to determining ensemble properties from FRET data, in which the polymer scaling exponent is allowed to vary with solution conditions. In its simplest form, it requires either the mean FRET efficiency or fluorescence lifetime information. In order to test the accuracy of the method, we have utilized both synthetic FRET data from implicit and explicit solvent simulations for 30 different protein sequences, and experimental single-molecule FRET data for an intrinsically disordered and a denatured protein. In all cases, we find that the inferred radii of gyration are within 10% of the true values, thus providing higher accuracy than simpler polymer models. In addition, the scaling exponents obtained by our procedure are in good agreement with those determined directly from the molecular ensemble. Our approach can in principle be generalized to treating other ensemble-averaged functions of intramolecular distances from experimental data.

Evidence for proton tunneling and a transient covalent flavin-substrate adduct in choline oxidase S101A

The effect of temperature on the reaction of alcohol oxidation catalyzed by choline oxidase was investigated with the S101A variant of choline oxidase. Anaerobic enzyme reduction in a stopped-flow spectrophotometer was biphasic using either choline or 1,2-[2H4]-choline as a substrate. The limiting rate constants klim1 and klim2 at saturating substrate were well separated (klim1/klim2>9), and were >15-fold slower than for wild-type choline oxidase. Solvent deuterium kinetic isotope effects (KIEs) ~4 established that klim1 probes the proton transfer from the substrate hydroxyl to a catalytic base. Primary substrate deuterium KIEs ≥7 demonstrated that klim2 reports on hydride transfer from the choline alkoxide to the flavin. Between 15°C and 39°C the klim1 and klim2 values increased with increasing temperature, allowing for the analyses of H+ and H– transfers using Eyring and Arrhenius formalisms. Temperature-independent KIE on the klim1 value (H2Oklim1/D2Oklim1) suggests that proton transfer occurs within a highly reorganized tunneling-ready-state with a narrow distribution of donor-acceptor distances. Eyring analysis of the klim2 value gave lines with the slope(choline)>slope(D-choline), suggesting kinetic complexity. Spectral evidence for the transient occurrence of a covalent flavin-substrate adduct during the first phase of the anaerobic reaction of S101A CHO with choline is presented, supporting the notion that an important role of amino acid residues in the active site of flavin-dependent enzymes is to eliminate alternative reactions of the versatile enzyme-bound flavin for the reaction that needs to be catalyzed.

Joint refinement of FRET measurements using spectroscopic and computational tools

The variability of the orientation factor is a long-standing challenge in converting FRET efficiency measurements into donor-acceptor distances. We propose the use of molecular dynamics (MD) simulations to characterize orientation distributions and thus improve the accuracy of distance measurements. Here, we test this approach by comparing experimental and simulated FRET efficiencies for a model donor-acceptor pair of enhanced cyan and enhanced yellow FPs connected by a flexible linker. Several spectroscopic techniques were used to characterize FRET in solution. In addition, a series of atomistic MD simulations of a total length of 1.5 μs were carried out to calculate the distances and the orientation factor in the FRET-pair. The resulting MD-based and experimentally measured FRET efficiency histograms coincided with each other, allowing for direct comparison of distance distributions. Despite the fact that the calculated average orientation factor was close to 2/3, the application of the average κ2 to the entire histogram of FRET efficiencies resulted in a substantial artificial broadening of the calculated distribution of apparent donor-acceptor distances. By combining single pair-FRET measurements with computational tools, we demonstrate that accounting for the donor and acceptor orientation heterogeneity is critical for accurate representation of the donor-acceptor distance distribution from FRET measurements.

Conformational States of Cytochrome P450 Oxidoreductase Evaluated by Förster Resonance Energy Transfer Using Ultrafast Transient Absorption Spectroscopy

NADPH-cytochrome P450 oxidoreductase (CYPOR) was shown to undergo large conformational rearrangements in its functional cycle. Using a new Förster resonance energy transfer (FRET) approach based on femtosecond transient absorption spectroscopy (TA), we determined the donor-acceptor distance distribution in the reduced and oxidized states of CYPOR. The unmatched time resolution of TA allowed the quantitative assessment of the donor-acceptor FRET, indicating that CYPOR assumes a closed conformation in both reduced and oxidized states in the absence of the redox partner. The described ultrafast TA measurements of FRET with readily available red-infrared fluorescent labels open new opportunities for structural studies in chromophore-rich proteins and their complexes.

Proton-Coupled Electron Transfer in a Series of Ruthenium-Linked Tyrosines with Internal Bases: Evaluation of a Tunneling Model for Experimental Temperature-Dependent Kinetics

Photoinitiated proton-coupled electron transfer (PCET) kinetics has been investigated in a series of four modified tyrosines linked to a ruthenium photosensitizer in acetonitrile, with each tyrosine bearing an internal hydrogen bond to a covalently linked pyridine or benzimidazole base. After correcting for differences in driving force, it is found that the intrinsic PCET rate constant still varies by 2 orders of magnitude. The differences in rates, as well as the magnitude of the kinetic isotope effect (KIE = kH/kD), both generally correlate with DFT calculated proton donor-acceptor distances. An Arrhenius analysis of temperature dependent data shows that the difference in reactivity arises primarily from differences in activation energies. We use this kinetic data to evaluate a commonly employed theoretical model for proton tunneling which includes a harmonic distribution of proton donor-acceptor distances due to vibrational motions of the molecule. Applying this model to the experimental data yields the conclusion that donor-acceptor compression is more facile in the compounds with shorter PT distance; however, this is contrary to independent calculations for the same compounds. This discrepancy is likely because the assumption in the model of Morse-shaped proton potential energy surfaces is inappropriate for (strongly) hydrogen-bonded systems. These results question the general applicability of this model. The results also suggest that a correlation of rate vs proton tunneling distance for the series of compounds is complicated by a concomitant variation of other relevant parameters.

Effect of Diffusion on Resonance Energy Transfer Rate Distributions: Implications for Distance Measurements.

Intrinsically disordered protein regions and many other biopolymers lack the three-dimensional structure that could be determined by X-ray crystallography or NMR, which encourages the application of alternative experimental methods. Time-resolved resonance energy transfer data are often used to measure distances between two fluorophores attached to a flexible biopolymer. This is complicated by the rotational and translational diffusion of the fluorophores and by nonmonoexponential donor decay in the absence of the acceptor. Equation I(DA)(t) = I(D)(t)·F(t) is derived here, which is applicable regardless of whether I(D)(t) is monoexponential. I(D)(t) and I(DA)(t) are the δ-excitation donor emission decays in the absence and in the presence of the acceptor; F(t) contains information about energy transfer, donor-acceptor distance distribution, and diffusion dynamics. It is shown that in the absence of rotational and translational diffusion, F(t) is a continuous distribution of exponentials, whereas in the presence of rotational and translational diffusion, F(t) is a sum of discrete exponentials. For each case it is shown how F(t) is related to the distance distribution. Experimental data obtained with a flexible tetradecapeptide in aqueous solution clearly demonstrate that F(t) is a sum of discrete exponential terms. A partial differential equation describing resonance energy transfer in the presence of both rotational and translational diffusion of the donor and acceptor tethered to the ends of a semiflexible chain is solved in this work using a combination of analytical and numerical methods; the solution is used to fit time-resolved emission of the donor, which makes it possible to determine the model parameters: contour length, persistence length, and the end-to-end translational diffusion coefficient.

Maximum Entropy Method-Based Acceptor Triplet-State Fluorescence Correlation Spectroscopy Analysis for Determination of Donor–Acceptor Distance Distribution: Theory and Simulation

A maximum entropy method-based acceptor triplet-state fluorescence correlation spectroscopy (tsFCS) analysis, i.e., tsFCS fluorescence resonance energy transfer (tsFCS-FRET), was proposed to resolve the donor-acceptor distance (R) distribution of a FRET system with multiple distances. An R-dependent acceptor triplet-state weight distribution function was introduced into the excited tsFCS model with a fixed R, and the weight distribution maximized the value of the Shannon entropy. tsFCS-FRET analysis showed consistent distributions with the actual pre-input for both unimodal distribution and two-species systems and a distribution with three peaks close to the pre-inputted values for three-species system at higher donor laser power. Collectively, tsFCS-FRET provides a powerful tool for resolving the R distribution, in turn, to the FRET efficiency (E) distribution, of a FRET system containing multiple E species.

Picosecond-resolved solvent reorganization and energy transfer in biological and model cavities

Water molecules in hydrophobic biological cleft/cavities are of contemporary interest for the biomolecular structure and molecular recognition of hydrophobic ligands/drugs. Here, we have explored picosecond-resolved solvation dynamics of water molecules and associated polar amino acids in the hydrophobic cleft around Cys-34 position of Endogenous Serum Albumin (ESA). While site selective acrylodan labeling to Cys-34 allows us to probe solvation in the cleft, Förster resonance energy transfer (FRET) from intrinsic fluorescent amino acid Trp 214 to the extrinsic acrylodan probes structural integrity of the protein in our experimental condition. Temperature dependent solvation in the cleft clearly shows that the dynamics follows Arrhenius type behavior up to 60 °C, after which a major structural perturbation of the protein is evident. We have also monitored polarization gated dynamics of the acrylodan probe and FRET from Trp 214 to acrylodan at various temperatures. The dynamical behavior of the immediate environments around the probe acrylodan in the cleft has been compared with a model biomimetic cavity of a reverse micelle (w0 = 5). Using same fluorescent probe of acrylodan, we have checked the structural integrity of the model cavity at various temperatures using picosecond-resolved FRET from Trp to acrylodan in the cavity. We have also estimated possible distribution of donor-acceptor distances in the protein and reverse micelles. Our studies reveal that the energetics of the water molecules in the biological cleft is comparable to that in the model cavity indicating a transition from bound state to quasibound state, closely consistent with a recent MD simulation study.

Probing Conformational Changes and Dynamics in eIF4A Helicase during RNA Unwinding by Single-Molecule FRET

The translation initiation factor eIF4A is the prototypical DEAD-box RNA helicase and it has a dumbbell structure consisting of two domains connected by a linker. Our previous studies have shown that eIF4A/eIF4H complex can bind directly to loop structures and repetitively unwind the RNA hairpin. To further illuminate the conformation change of eIF4A during RNA unwinding, we label several well chosen mutant eIF4A with donor and acceptor and encapsulate eIF4A and RNA substrate within lipid vesicles. The cotrapping of eIF4A and RNA is detected by multicolor colocalization and the interdomain and intradomain distance change of eIF4A is investigated by single-molecule Fluorescence Resonance Energy Transfer (sm-FRET). First we show that the linker which connects the two domain is very flexible by showing broad donor-acceptor distance distribution. Second we demonstrate that eIF4A in the presence of eIF4H can repetitively unwind the RNA hairpin substrate by undergoing the open-and-closed conformation using the energy from ATP hydrolysis. And the interdomain interaction is mainly responsible for the helicase activity. Furthermore, we studied how RNA aptamer and small molecule, hippuristanol, interact with eIF4A and change its conformation. These studies can further illuminate the molecular mechanism how they inhibit the helicase activity of eIF4A.

Effects of the Donor–Acceptor Distance and Dynamics on Hydride Tunneling in the Dihydrofolate Reductase Catalyzed Reaction

A significant contemporary question in enzymology involves the role of protein dynamics and hydrogen tunneling in enhancing enzyme catalyzed reactions. Here, we report a correlation between the donor-acceptor distance (DAD) distribution and intrinsic kinetic isotope effects (KIEs) for the dihydrofolate reductase (DHFR) catalyzed reaction. This study compares the nature of the hydride-transfer step for a series of active-site mutants, where the size of a side chain that modulates the DAD (I14 in E. coli DHFR) is systematically reduced (I14V, I14A, and I14G). The contributions of the DAD and its dynamics to the hydride-transfer step were examined by the temperature dependence of intrinsic KIEs, hydride-transfer rates, activation parameters, and classical molecular dynamics (MD) simulations. Results are interpreted within the framework of the Marcus-like model where the increase in the temperature dependence of KIEs arises as a direct consequence of the deviation of the DAD from its distribution in the wild type enzyme. Classical MD simulations suggest new populations with larger average DADs, as well as broader distributions, and a reduction in the population of the reactive conformers correlated with the decrease in the size of the hydrophobic residue. The more flexible active site in the mutants required more substantial thermally activated motions for effective H-tunneling, consistent with the hypothesis that the role of the hydrophobic side chain of I14 is to restrict the distribution and dynamics of the DAD and thus assist the hydride-transfer. These studies establish relationships between the distribution of DADs, the hydride-transfer rates, and the DAD's rearrangement toward tunneling-ready states. This structure-function correlation shall assist in the interpretation of the temperature dependence of KIEs caused by mutants far from the active site in this and other enzymes, and may apply generally to C-H→C transfer reactions.

A Comparison of Donor-Acceptor Distance Distributions Derived from Ensemble and Single-Pair FRET Measurements

We engineered a FRET system consisting of a DNA oligonucleotide with FRET acceptor AlexaFluor647 at the 3′ end and a downstream DNA oligonucleotide with FRET donor Cy3 at the 5′ end. These were annealed to antisense DNA oligonucleotide that provided a four nucleotide gap between donor and acceptor residues. Molecular modeling placed the mean Forster radius-normalized inter-probe distance r/R0 between 0.5 and 1.Transfer efficiency was estimated from steady state fluorescence (ssFRET), ensemble time-resolved fluorescence (en-trFRET), and single-pair time resolved fluorescence (sp-trFRET). In our analysis, we sought internal consistency in the estimated FRET efficiency across the measurements. A distribution of inter-probe distances was obtained from the en-trFRET data after including contributions from scattering and unquenched donor in the analysis. Similarly, a distribution of inter-probe distance was obtained from the sp-trFRET data after accounting for scattering by passing the time-tagged time-resolved (TTTR) data through a time-correlated single photon counting (TCSPC)-based time gated filter and accounting for the population of unquenched donor by collecting data using pulsed interleaved excitation (PIE). We compare the recovered distance distributions from en-trFRET and sp-trFRET.

Resonance Energy-Transfer Studies of the Conformational Change on the Adsorption of Oligonucleotides to a Silica Interface

Time-resolved evanescent wave-induced fluorescence studies have been carried out on a series of fluorescently labeled oligonucleotide sequences adsorbed to a silica surface from solution. The fluorescence decay profiles of a fluorescent energy donor group undergoing resonance energy transfer to a nonemissive energy-acceptor molecule have been analyzed in terms of a distribution of donor-acceptor distances to reveal the conformational changes that occur in these oligonucleotides upon adsorption. Evanescent wave-induced time-resolved Förster resonance energy-transfer (EW-TRFRET) measurements indicate that at a high electrolyte concentration, there is localized separation of the oligonucleotide strands, and the helical structure adopts an "unraveled" conformation as a result of adsorption. This is attributed to the flexibility within the oligonucleotide at high electrolyte concentration allowing multiple segments of the oligonucleotide to have direct surface interaction. In contrast, the EW-TRFRET measurements at a lower electrolyte concentration reveal that the oligonucleotide retains its helical conformation in a localized extended state. This behavior implies that the rigidity of the oligonucleotide at this electrolyte concentration restricts direct interaction with the silica to a few segments, which correspondingly introduces kinks in the double helix conformation and results in significant oligonucleotide segmental extension into solution.

Protein Structure and Dynamics from Single-Molecule Fluorescence Resonance Energy Transfer

The pros and cons of single-molecule vs ensemble-averaged fluorescence resonance energy transfer (FRET) experiments, performed on proteins, are explored with the help of Langevin dynamics simulations. An off-lattice model of the polypeptide chain is employed, which gives rise to a well-defined native state and two-state folding kinetics. A detailed analysis of the distribution of the donor-acceptor distance is presented at different points along the denaturation curve, along with its dependence on the averaging time window. We show that unique information on the correlation between structure and dynamics, which can only be obtained from single-molecule experiments, is contained in the correlation between the donor-acceptor distance and its displacement. The latter is shown to provide useful information on the free energy landscape of the protein, which is complementary to that obtained from the distribution of donor-acceptor distances.

Time‐resolved fluorescence resonance energy transfer: A versatile tool for the analysis of nucleic acids

The biological functions of nucleic acids in processes of DNA replication, transcription, homologous recombination, mRNA translation, and ribozyme catalysis are intimately linked to their three-dimensional structures and to conformational changes induced by proteins, metal ions and other ligands. Fluorescence spectroscopy is a powerful technique for probing the structure and conformational dynamics of biological macromolecules under a wide range of solution conditions. Fluorescence resonance energy transfer (FRET) provides long-range distance information from 10 to 100 Å, a range that is useful for probing the global structure of nucleic acids. While steady-state measurements of FRET provide the average distance between donor and acceptor, much more information is available from the analysis of the nanosecond emission decay of the donor in time-resolved FRET (trFRET) experiments. Analysis of the decay in terms of donor–acceptor distance distributions can resolve different conformers in a heterogeneous mixture, providing information on the global structure and flexibility of each species as well as their equilibrium populations. In this review, we outline the principles of trFRET and the methods used to incorporate fluorescent probes into DNA and RNA. Examples of specific applications are presented to illustrate the versatility of trFRET as a tool to define global structures, to identify conformational heterogeneity and flexibility, to investigate the energetics of tertiary structure formation and to probe structural rearrangements of nucleic acids. © 2002 Wiley Periodicals, Inc. Biopoly (Nucleic Acid Sci) 61: 159–179, 2002

Time-resolved fluorescence resonance energy transfer: a versatile tool for the analysis of nucleic acids.

The biological functions of nucleic acids in processes of DNA replication, transcription, homologous recombination, mRNA translation, and ribozyme catalysis are intimately linked to their three-dimensional structures and to conformational changes induced by proteins, metal ions and other ligands. Fluorescence spectroscopy is a powerful technique for probing the structure and conformational dynamics of biological macromolecules under a wide range of solution conditions. Fluorescence resonance energy transfer (FRET) provides long-range distance information from 10 to 100 A, a range that is useful for probing the global structure of nucleic acids. While steady-state measurements of FRET provide the average distance between donor and acceptor, much more information is available from the analysis of the nanosecond emission decay of the donor in time-resolved FRET (trFRET) experiments. Analysis of the decay in terms of donor-acceptor distance distributions can resolve different conformers in a heterogeneous mixture, providing information on the global structure and flexibility of each species as well as their equilibrium populations. In this review, we outline the principles of trFRET and the methods used to incorporate fluorescent probes into DNA and RNA. Examples of specific applications are presented to illustrate the versatility of trFRET as a tool to define global structures, to identify conformational heterogeneity and flexibility, to investigate the energetics of tertiary structure formation and to probe structural rearrangements of nucleic acids.

Ultrafast excited-state energy migration dynamics in an efficient light-harvesting antenna polymer based on Ru(II) and Os(II) polypyridyl complexes.

A detailed study of the excited state energy migration dynamics that take place within an assembly of Ru(II) and Os(II) polypyridyl complexes linked together through a polymer backbone is presented. The energy migration process is initiated by the photoexcitation of the metal-to-ligand charge transfer (MLCT) transition in one of the Ru(II) complexes and terminated by energy transfer to a lower energy Os(II) trap. Energy transfer sensitization of Os(II) can occur in a single step if the excited state is formed adjacent to a trap, or after a series of hops between isoenergetic rutheniums prior to reaching a trap. The dynamics of the energy transfer process are followed by monitoring the growth of Os(II) luminescence at 780 nm. The kinetics of the growth are complex and can be fit by a sum of two exponentials. This kinetic complexity arises both from the presence of a distribution of donor-acceptor distances and the variety of time scales by which Os(II) can be formed. We have augmented the time-resolved experiments with Monte Carlo simulations, which provide insight into the polymer array's structure and at the same time form the basis of a molecular-level description of the energy migration dynamics. The simulations indicate that the most probable Ru-->Os energy transfer time is approximately 400 ps while the time scale for Ru-->Ru hopping is approximately 1-4 ns. The time scale for Ru-->Ru hopping relative to its natural lifetime (1000 ns) suggests that this polymer system could be extended to considerably longer dimensions without an appreciable loss in its overall efficiency.

Influence of solvent and configuration of residues at positions 2 and 3 on distance and mobility of pharmacophore groups at positions 1 and 4 in cyclic enkephalin analogues

The analgesic activity of opioid peptides is mainly connected with their affinity and selectivity for the μ-receptors. The biological activity of cyclic opioid analogues depends on mutual orientation and conformational freedom of aromatic pharmacophore groups at positions 1 and 4. The distance and distance distributions between chromophores at positions 1 [Phe(p-NO2), p-nitrophenylalanine] and 4 [Nal, β-(2-naphthyl)alanine], which constitute an energy donor–acceptor pair, were calculated based on measured fluorescence intensity decays of a donor (Nal). The influence of the solvent and configuration of the residues at position 2 and 3 on donor–acceptor distance distribution and mobility of pharmacophore groups at position 1 and 4 in cyclic enkephalin analogues are discussed. © 2001 John Wiley & Sons, Inc. Biopolymers 59: 180–190, 2001