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Guided dental implant surgeries, their associated stackable surgical guides, and respective prosthetic techniques facilitate control and precision during implant placement and simultaneously streamline immediate provisionalization. Although implant treatments have demonstrated documented success, their failures both immediately and over time have traditionally required an additional surgery, a completely new prosthetic work-up, and subsequent fabrication of associated surgical placement components. This article describes a routine to recover the original implant placement treatment plan, without the added time, expense, or inconvenience involved in redeveloping the implant placement surgical guide. A case is presented to document the protocol for this routine.

Stark spectroscopy has been an invaluable tool to reveal information about the physicochemical properties of a molecule, specifically its dielectric properties or charge redistribution [1] . Typically, two molecular properties dominate Stark spectra, i.e. a dipole moment change or a difference in polarizability between ground and excited state. An analysis of the Stark effect leads to a deeper understanding of photo-induced electron and charge transfer, biological organization and energy tuning, and design of nonlinear material properties [2] - [3] . However, a successful measurement in a conventional system, which employs a low frequency alternating current (AC) voltage source, is subject to some preconditions such as limited sample thickness, limited E-field strength, geometric constraints, and freezing issues. To overcome such limitations, we demonstrate terahertz (THz) Stark spectroscopy. Using intense single cycle THz pulses as the electric field source and monitoring transient spectral responses with a coincident fs-supercontinuum (SC) pulse offers advantages over traditional Stark spectroscopy and opens previously inaccessible perspectives. 1) THz pulses, with their picosecond transients, oscillate faster than typical solvent responses and consequently eliminate the requirement to freeze the samples to prevent poling effects. 2) THz frequencies allow for peak fields much beyond the dielectric breakdown limit in conventional low frequency experiments. 3) All-optical approaches eliminate the geometric issues of applying E-fields to different sample thicknesses in arbitrary polarization directions. Thus, we can now conduct time-resolved studies at room temperature and at ambient conditions more relevant to physiological or operative conditions.

Flavins are photoenzymatic cofactors often exploiting the absorption of light to energize photoinduced redox chemistry in a variety of contexts. Both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are used for this function. The study of these photoenzymes has been facilitated using flavin analogs. Most of these analogs involve modification of the flavin ring, and there is recent evidence that adenine (Ade)-modified FAD can affect enzyme turnover, but so far this has only been shown for enzymes where the adenine and flavin rings are close to each other in a stacked conformation. FAD is also stacked in aqueous solution, and its photodynamics are quite different from unstacked FAD or FMN. Oxidized photoexcited FAD decays rapidly, presumably through PET with Ade as donor and Fl* as acceptor. Definitive identification of the spectral signatures of Ade

International audience; Ultrafast optical control of intramolecular charge flow was demonstrated, which paves the way for photocurrent modulation and switching with a highly wavelength‐selective ON/OFF ratio. The system that was explored is a fac‐[Re(CO)3(TTF‐DPPZ)Cl] complex, where TTF‐DPPZ=4’,5’‐bis(propylthio)tetrathiafulvenyl[i]dipyrido[3,2‐a:2’,3’‐c]phenazine. DFT calculations and AC‐Stark spectroscopy confirmed the presence of two distinct optically active charge‐transfer processes, namely a metal‐to‐ligand charge transfer (MLCT) and an intra‐ligand charge transfer (ILCT). Ultrafast transient absorption measurements showed that the ILCT state decays in the ps regime. Upon excitation to the MLCT state, only a long‐lived 3MLCT state was observed after 80 ps. Remarkably, however, the bleaching of the ILCT absorption band remained as a result of the effective inhibition of the HOMO–LUMO transition.

Lumichrome (7,8-dimethylalloxazine, LC) is a natural photodegradation product and catabolite of flavin coenzymes. Although not a coenzyme itself, LC is used for biosignaling in plants and single-celled organisms, including quorum sensing in the formation of biofilms. The noninvasive detection of in vivo lumichrome would be useful for monitoring this signaling event. For molecules that undergo significant charge redistribution upon light excitation (e.g., intramolecular charge transfer), there are optical detection methods (e.g., second-harmonic generation) that would be well suited to this task. Here, we have used Stark spectroscopy to measure the extent and direction of charge redistribution in photoexcited LC. Stark and low-temperature absorption spectra were obtained at 77 K on LC in ethanol glasses and analyzed using the Liptay analysis to obtain the difference dipole moments and polarizabilities. These data were complemented by a computational analysis of the excited states using density functional theory (DFT) at the TD-B3LYP/6-311+G(2d,p) level of theory.

Ultrafast optical control of intramolecular charge flow was demonstrated, which paves the way for photocurrent modulation and switching with a highly wavelength-selective ON/OFF ratio. The system that was explored is a fac-[Re(CO)(3)(TTF-DPPZ)Cl] complex, where TTF-DPPZ = 4’,5’-bis(propylthio)tetrathiafulvenyl[i]dipyrido[3,2-a:2’,3’-c]phenazine. DFT calculations and AC-Stark spectroscopy confirmed the presence of two distinct optically active charge-transfer processes, namely a metal-to-ligand charge transfer (MLCT) and an intra-ligand charge transfer (ILCT). Ultrafast transient absorption measurements showed that the ILCT state decays in the ps regime. Upon excitation to the MLCT state, only a long-lived (3)MLCT state was observed after 80 ps. Remarkably, however, the bleaching of the ILCT absorption band remained as a result of the effective inhibition of the HOMO–LUMO transition.

The annulation of two redox-active molecules into a compact and planar structure paves the way toward a new class of electronically versatile materials whose physical properties can be tuned via a substitution of one of the constituting moieties. Specifically, we present tetrathiafulvalene−benzothiadiazole donor−acceptor molecules. The critical role played by the dielectric properties of these molecules is evident by the large spectral shifts of the ground-state absorption spectra in a range of solvents. Stark spectroscopy is performed to determine experimentally dipole and polarizability change over transitions in the visible range with particular attention to the transition from the highest-occupied molecular orbital (HOMO) to the lowest-unoccupied molecular orbital (LUMO). The experimental results are compared to the results of time-dependent density functional theory calculations, and we reciprocally validate results from calculation and experiment. This allows us to filter out effective models and reveal important insights. The calculations are initially performed in the gas phase and subsequently a polarizable continuum model is adopted to probe the in fluence of the solvent on the molecular dielectric properties. The results show a large charge displacement from the HOMO to the LUMO and confirm the intramolecular charge transfer nature of the lowest-energy transition. Substitution of the acceptor moiety with electron-withdrawing groups results in changes to the experimentally determined molecular properties consistent with the effects predicted by computational results. The dominant contribution to the electroabsorption signal is due to the change in dipole moment, which is measured to be roughly 20 D for all samples and forms a small angle with the transition dipole moment in a toluene solvent environment.

Light-driven DNA repair by extremophilic photolyases is of tremendous importance for understanding the early development of life on Earth. The mechanism for flavin adenine dinucleotide repair of DNA lesions is the subject of debate and has been studied mainly in mesophilic species. In particular, the role of adenine in the repair process is poorly understood. Using molecular docking, molecular dynamics simulations, electronic structure calculations, and electron tunneling pathways analysis, we examined adenine’s role in DNA repair in four photolyases that thrive at different temperatures. Our results indicate that the contribution of adenine to the electronic coupling between the flavin and the cyclobutane pyrimidine dimer lesion to be repaired is significant in three (one mesophilic and two extremophilic) of the four enzymes studied. Our analysis suggests that thermophilic and hyperthermophilic photolyases have evolved structurally to preserve the functional position (and thus the catalytic function) of adenine at their high temperatures of operation. Water molecules can compete with adenine in establishing the strongest coupling pathway for the electron transfer repair process, but the adenine contribution remains substantial. The present study also reconciles prior seemingly contradictory conclusions on the role of adenine in mesophile electron transfer repair reactions, showing how adenine-mediated superexchange is conformationally gated.

Radical pair formation and decay are implicated in a wide range of biological processes including avian magnetoreception. However, studying such biological radical pairs is complicated by both the complexity and relative fragility of natural systems. To resolve open questions about how natural flavin–amino acid radical pair systems are engineered, and to create new systems with novel properties, we developed a stable and highly adaptable de novo artificial protein system. These protein maquettes are designed with intentional simplicity and transparency to tolerate aggressive manipulations that are impractical or impossible in natural proteins. Here we characterize the ultrafast dynamics of a series of maquettes with differing electron-transfer distance between a covalently ligated flavin and a tryptophan in an environment free of other potential radical centers. We resolve the spectral signatures of the cysteine-ligated flavin singlet and triplet states and reveal the picosecond formation and recombination of singlet-born radical pairs. Magnetic field-sensitive triplet-born radical pair formation and recombination occurs at longer timescales. These results suggest that both triplet- and singlet-born radical pairs could be exploited as biological magnetic sensors.

Chromophoric biomolecules are exploited as reporters of a diverse set of phenomena, acting as internal distance monitors, environment and redox sensors, and endogenous imaging probes. The extent to which they can be exploited is dependent on an accurate knowledge of their fundamental electronic properties. Arguably of greatest importance is a precise knowledge of the direction(s) of the absorption transition dipole moment(s) (TDMs) in the molecular frame of reference. Such is the case for flavins, fluorescent redox cofactors utilized for ground and excited-state redox and photochemical processes. The directions of the TDMs in oxidized and semiquinone flavins were characterized decades ago, and the details of charge redistribution in these forms have also been studied by Stark spectroscopy. The electronic structure of the fully reduced hydroquinone anionic state, FlH(−), however, has been the subject of unfounded assumptions and estimates about the number and direction of TDMs in FlH(−), as well the electronic structure changes that occur upon light absorption. Here we have used Stark spectroscopy to measure the magnitude and direction of charge redistribution in FlH(−) upon optical excitation. These data were analyzed using TD-DFT calculations. The results show unequivocally that not one but two nearly orientation-degenerate electronic transitions are required to explain the 340–500 nm absorption spectral range, demolishing the commonly held assumption of a single transition. The difference dipole moments for these states show that electron density shifts toward the xylene ring for both transitions. These measurements force a reappraisal of previous studies that have used erroneous assumptions and unsubstantiated estimates of these quantities. The results put future optical studies of reduced flavins/flavoproteins on a firm photophysical footing.

DNA photolyase has been the topic of extensive studies due to its important role of repairing photodamaged DNA, and its unique feature of using light as an energy source. A crucial step in the repair by DNA photolyase is the forward electron transfer from its cofactor (FADH(-) ) to the damaged DNA, and the detailed mechanism of this process has been controversial. In the present study, we examine the forward electron transfer in DNA photolyase by carrying out high-level ab initio calculations in combination with a quantum mechanical/molecular mechanical (QM/MM) approach, and by measuring fluorescence emission spectra at low temperature. On the basis of these computational and experimental results, we demonstrate that multiple decay pathways exist in DNA photolyase depending on the wavelength at excitation and the subsequent transition. This implies that the forward electron transfer in DNA photolyase occurs not only by superexchange mechanism but also by sequential electron transfer.

The optical spectrum of a flavoprotein is one of its signature properties. No two flavoprotein absorption spectra are exactly alike as each encodes the details of the interaction of the flavin cofactor electronic structure with the specific protein binding pocket. Electronic Stark spectroscopy has the potential to elucidate these interactions with high sensitivity, at low cost, and requiring minimal technical sophistication. In this chapter we will outline the theoretical basis for Stark spectroscopy and describe the construction of the Stark spectrometer. Step-by-step instructions are given for acquiring and interpreting Stark spectra to retrieve difference moments of the flavin ground versus excited state charge distributions.

The phrB gene encoding a putative cold-adapted DNA photolyase was cloned from the bacterial genomic DNA of Colwellia psychrerythraea 34H, a psychrophilic bacterium. Recombinant DNA photolyase, rCpPL, was overexpressed and purified from three different vectors. rCpPL binds its DNA substrate by flipping a cyclobutane pyrimidine dimer (CPD) into its active site and repairs CPD-containing DNA in vitro. rCpPL contains one catalytic flavin adenine dinucleotide (FAD) cofactor, but displays promiscuity in cofactor binding, in which either a flavin mononucleotide (FMN) or a methenyltetrahydrofolate (MTHF) molecule is bound as an antenna molecule and found in sub-stoichiometric amounts. The UV/Vis spectrum of oxidized rCpPL shows that the FADOX absorption maximum is the most red-shifted reported for a PL, suggesting a unique cavity electrostatic environment. Modest FAD vibronic structure suggests that the binding pocket is more flexible than warmer PLs, corroborating the hypothesis that psychrophilic proteins must be highly flexible to function at low temperatures. Fluorescence excitation data show that the freshly purified flavin cofactor is in its fully reduced state (FADH¯). A homology analysis of PL protein structures spanning 70 °C in growth temperature supports the data that the structure of CpPL is quite different from its warmer cousins.

Reduced anionic flavin adenine dinucleotide (FADH(−)) is the critical cofactor in DNA photolyase (PL) for the repair of cyclobutane pyrimidine dimers (CPD) in UV-damaged DNA. The initial step involves photoinduced electron transfer from *FADH(−) to the CPD. The adenine (Ade) moiety is nearly stacked with the flavin ring, an unusual conformation compared to other FAD-dependent proteins. The role of this proximity has not been unequivocally elucidated. Some studies suggest that Ade is a radical intermediate, but others conclude that Ade modulates the electron transfer rate constant (k(ET)) through superexchange. No study has succeeded in removing or modifying this Ade to test these hypotheses. Here, FAD analogs containing either an ethano- or etheno-bridged Ade between the AN1 and AN6 atoms (e-FAD and ε-FAD, respectively) were used to reconstitute apo-PL, giving e-PL and ε-PL respectively. The reconstitution yield of e-PL was very poor, suggesting that the hydrophobicity of the ethano group prevented its uptake, while ε-PL showed 50% reconstitution yield. The substrate binding constants for ε-PL and rPL were identical. ε-PL showed a 15% higher steady-state repair yield compared to FAD-reconstituted photolyase (rPL). The acceleration of repair in ε-PL is discussed in terms of an ε-Ade radical intermediate vs superexchange mechanism.

DNA photolyase can be used to study how a protein with its required cofactor has adapted over a large temperature range. The enzymatic activity and thermodynamics of substrate binding for protein from Sulfolobus solfataricus were directly compared to protein from Escherichia coli. Turnover numbers and catalytic activity were virtually identical, but organic cosolvents may be necessary to maintain activity of the thermophilic protein at higher temperatures. UV-damaged DNA binding to the thermophilic protein is less favorable by ∼2 kJ/mol. The enthalpy of binding is ∼10 kJ/mol less exothermic for the thermophile, but the amount and type of surface area buried upon DNA binding appears to be somewhat similar. The most important finding was observed when ionic strength studies were used to separate binding interactions into electrostatic and nonelectrostatic contributions; DNA binding to the thermophilic protein appears to lack the electrostatic contributions observed with the mesophilic protein.

8-Vinyladenosine (8VA) is an adenine analog that effectively base pairs with thymine in duplex DNA. With a red-shifted absorption band centered around 290 nm excited 8VA emits Stokes-shifted fluorescence centered at 370 nm. The fluorescence quantum yield of 8VA* is sensitive to base stacking, making it an effective reporter of DNA structure and dynamics. We have performed cyclic voltammetry (CV) studies on 8VA in aprotic organic solvents to explore the mechanism of fluorescence quenching. Excited state oxidation and reduction potentials were calculated from the experimentally determined ground state one electron redox potentials. The Rehm–Weller equation was used to obtain the free energies of electron transfer for native nucleobase monophosphate oxidation and reduction paired with 8VA*. The magnitude and sign of the free energies have been used to assess whether electron transfer is thermodynamically plausible. The results suggest that electron transfer to or from 8VA* may not be the dominant mechanism of quenching for AMP, CMP, and perhaps GMP. In contrast, TMP may be an effective electron acceptor. Taken together, the results suggest that 8VA* quenching by nucleobase monophosphates in aqueous solution proceeds through a different mechanism than ET.

6-Methylisoxanthopterin (6-MI) is a pteridine-based guanine analog that has a red-shifted absorption and high fluorescence quantum yield. Its Watson-Crick base-pairing and base stacking properties are similar to guanine. The fluorescence quantum yield of 6-MI is sensitive to its nearest neighbors and base stacking, making it a very useful real-time probe of DNA structure. The fundamental photophysics underlying this fluorescence quenching by base stacking is not well understood. We have explored the excited-state electronic structure of the 6-MI in frozen 77 K LiCl glasses using Stark spectroscopy. These measurements yielded the direction and degree of charge redistribution for the S(0)→S(1) transition as manifested in the difference dipole moment, Δμ(01), and difference static polarizability, TrΔα. TDDFT (time-dependent density functional theory) was employed to calculate the transition energy, oscillator strength, and the dipole moments of the ground and lowest optically bright excited state of 6-MI (S(0)→S(1)). The direction of Δμ(01) was assigned in the molecular frame based on the Stark data and calculations. These results suggest that the C4═O and C2-NH(2) groups are electron-deficient in the excited state, a very different outcome compared with guanine. This implies that Watson-Crick hydrogen bonding in 6-MI may be modulated by absorption of a photon so as to strengthen base pairing, if only transiently. Solvatochromism was also obtained for the absorption and emission spectra of 6-MI in various solvents and compared with the Stark spectroscopic results using both the Lippert-Mataga and Bakhshiev models.

Fluorescent nucleic acid base analogues (FBAs) are used widely as probes of DNA and RNA structure and dynamics. Of increasing utility are the pteridone adenosine analogues (6MAP, DMAP) and pteridine guanosine analogues (3MI, 6MI). These FBAs (collectively referred to as PTERs) are useful, in part, because their fluorescence quantum yields, Phi(f), are modulated by base stacking with native bases (NBs), making them sensitive reporters of DNA structure. The quenching mechanism has been hypothesized to be photoinduced electron transfer following selective excitation of the FBA, but hard evidence for this has been lacking. The degree of quenching shows some dependence on the neighboring bases, but there has been no real determination as to whether FBA*:NB complexes satisfy the basic thermodynamic requirement for spontaneous PET: a negative free energy for the electron transfer reaction. Indeed, quenching may result from entirely different mechanisms. To address these questions, Stern-Volmer (S-V) experiments were performed using the native-base monophosphate nucleotides (NMPs) GMP, AMP, CMP, and dTMP in aqueous solutions as quenchers to obtain quenching rate constants, k(q). Cyclic voltammetry (CV) and optical absorption and emission data of the PTERS were obtained in aprotic organic solvents. These data were used to obtain excited-state redox potentials from which electron transfer free energies were derived using the Rehm-Weller equation. The reorganization energies for PET were obtained using the Scandola-Balzani equation, taking into account the free energy contribution due to water. 6MAP*, DMAP*, and 3MI* gave negative free energies between -0.1 and -0.2 eV and reorganization energies of about 0.13 eV. They all displayed ET activation energies below the accessible thermal energy (0.038 eV = 3/2k(B)T, where k(B) is Boltzmann's constant) for all NMPs with the exception of CMP, whose activation barrier was only about 35% higher (approximately 0.05 eV). Thus, we conclude that these PTERs act as electron acceptors and promote NMP oxidation. However, 6MI* had positive ET free energies for all NMPs with the exception of GMP (and then only for nucleobase oxidation). The magnitudes of these free energies (or = 0.45 eV for AMP, CMP, and dTMP) suggest that 6MI* may not quenched by PET.

The electronic structure of the two lowest excited electronic states of FAD and FADH(*) in folate-depleted E. coli DNA photolyase (PL(OX) and PL(SQ), respectively) was measured using absorption Stark spectroscopy. The experimental analysis was supported by TDDFT calculations of both the charge redistribution and the difference dipole moments for the transitions of both oxidation states using lumiflavin as a model. The difference dipole moments and polarizabilities for PL(OX) are similar to those obtained in our previous work for flavins in simple solvents and in an FMN-containing flavoprotein. No such comparison can be made for PL(SQ), as we believe this to be the first experimental report of the direction and magnitude of excited-state charge redistribution in any flavosemiquinone. The picture that emerges from these studies is discussed in the context of electron transfer in photolyase, particularly for the semiquinone photoreduction process, which involves nearby tryptophan residues as electron donors. The direction of charge displacement derived from an analysis of the Stark spectra rationalizes the positioning of the critical Trp382 residue relative to the flavin for efficient vectorial electron transfer leading to photoreduction. The ramifications of vectorial charge redistribution are discussed in the context of the wider class of flavoprotein blue light photoreceptors.

Cyclobutylpyrimidine dimers (CPDs) are the major UV photoproduct formed in DNA containing adjacent pyrimidines. These lesions can be repaired by DNA photolyase, a flavoprotein that utilizes blue light in a direct reversal of the cyclobutane ring. Previous studies have shown that the CPD is base flipped into the protein, with concomitant disruption of the substrate around the CPD. In this study, we use a fluorescent cytidine analog, pyrrolo-dC (PC), to probe how far base flipping propagates along the duplex. From these measurements, the degree of base destacking in the two bases flanking the adenines opposing the CPD appears to be minimal, which was consistent with the protein:substrate crystal structure. Fluorescence-detected melting temperatures for duplexes with and without a CPD were obtained, suggesting that a 5'-pyrimidine-PC-purine-3' motif is more stable than the 5'-purine-PC-pyrimidine-3' motif. This stability trend was reflected in the fluorescence intensities of ss-PC oligos but not for duplexes. The melting point depression due to the PC probe was found to be comparable to other popular fluorescent base analogs.

Cyclobutylpyrimidine dimers (CPDs) are formed between adjacent pyrimidines in DNA when it is exposed to ultraviolet light. CPDs can be directly repaired by DNA photolyase (PL) upon absorption of blue-green light. We have used the fluorescent adenine analogue 2-aminopurine (2Ap) to probe the local double-helical structure of the DNA substrate when it binds to the protein. Duplex melting temperatures and van't Hoff enthalpies were obtained by both UV-vis absorption and fluorescence spectroscopies to ascertain the effect of the probe and CPD on DNA stability. Steady-state fluorescence measurements of the single- and double-stranded oligos showed that the local region around the 5'-side of the CPD lesion was more disrupted and destacked than the 3'-side in substrate-protein complexes. These results were compared with those of a protein-substrate crystal structure, demonstrating that the crystal structure and solution-state studies are in agreement with regard to the differential distortions of the target DNA at the active site of the protein.

DNA photolyase (PL) is a monomeric flavoprotein that repairs cyclobutylpyrimidine dimers (CPDs) via photoinduced electron transfer from a reduced flavin adenine dinucleotide cofactor (FADH(-)) to the bound CPD. We have used subpicosecond UV transient absorption spectroscopy to measure the electron-transfer and repair kinetics of Anacystis nidulans DNA photolyase with dimeric and pentameric oligothymidine substrates. Here we show that the electron-transfer lifetime is 32 +/- 20 ps for the pentameric substrate. Repair of the carbon-carbon double bonds (C=C) in the CPD is initiated in approximately 60 ps, and bond scission appears to be completed by 1500 ps. This suggests that the repair of the two C=C bonds proceeds sequentially and that the first bond scission has a much lower activation barrier than the second. Our experiments also suggest that the semiquinone FADH(*) cofactor is not reduced to its catalytically active FADH(-) state by substrate after repair but remains in the semiquinone state. In contrast to the longer substrate, the dinucleotide substrate produced a mixture of kinetics representing bound and unbound substrate.

Low-temperature absorption and Stark spectroscopy have been used to study the electronic properties of oxidized flavin mononucleotide (FMN) in old yellow enzyme (OYE) and OYE complexed with p-chlorophenol (p-Cl phenol). The low-temperature absorbance spectrum of OYE showed splittings of the blue and near-UV vibronic bands, which appears to be due to hydrogen bonding between the isoalloxazine moiety and the protein. A Stark spectroscopic analysis showed that the electronic structure of the FMN cofactor in OYE is not significantly perturbed relative to flavins in simple solvents. However, the charge-transfer band in the OYE:p-Cl phenol complex showed a large Stark effect indicative of substantial charge displacement. The magnitude and direction of this charge displacement are consistent with significant charge transfer along the charge-transfer transition dipole moment direction. In addition, the Stark spectrum of the CT band showed unexpected fine structure that could correlate with vibrational progressions in either the p-Cl phenol donor or the flavin acceptor.

Adaptation to extreme environments is a seminal characteristic of life on Earth. Nowhere is this property more strongly evident than in single-celled organisms. Bacteria, archaea, and eukaryotes all have representatives that thrive in cold or hot environments. Many of these extremophile enzymes share significant amino acid sequence homology in spite of very different optimal growth temperatures (TG). Based on a simple amino acid sequence analysis it is unclear what adaptive changes are required to tailor homologous enzymes to function in extreme environments.A case in point is DNA photolyase (PL), a monomeric flavoprotein that binds UV-damaged DNA and repairs it by blue light activated picosecond electron transfer from a conserved flavin adenine dinucleotide cofactor (FADH¯) to the tightly bound cyclobutylpyrimidine dimer DNA lesion. These enzymes also include a second light-harvesting cofactor. Here we present a comparative analysis of three recombinant CPD photolyases, hyperthermophilic Sulfolobus solfataricus (TG=353K, rSsPL), mesophilic E. coli (TG=310K, rEcPL), and psychrophilic Colwellia psychrerythraea (TG=281K, rCpPL).All PLs utilize FADH¯ bound in a highly conserved site for repair. In addition, each PL demonstrates different properties for its 2nd cofactor. We used a variety of biochemical, physical, and molecular biological tools to compare these extremophile proteins with regard to repair yield, cofactor reduction potential and excited state properties. The difference in the stability of the redox state of the purified protein suggests different structural adaptations of each PL to their respective thermal environments. Denaturation studies reveal that rSsPL has a very stable structure, whereas rCpPL is extremely sensitive to its thermal and aqueous environments. A comparison of cofactor absorption and emission spectra reveal significant differences in how the flavin cofactor is bound in the binding pocket, in spite of significant amino acid sequence homology for this cavity across all proteins.

A new theoretical model addresses the reduction in the quantum yield for primary charge separation in bacterial photosynthetic reaction centers (RCs) in an applied electric field. The electric field dependence of the internal conversion rate constant for the primary donor is proposed to act as a drain that competes with primary charge separation. The field dependence of the internal conversion process is based on experimental data that have shown that the excited state of the primary donor has a large polarizability suggesting that the excitonic basis of the primary donor is mixed with charge transfer (CT) basis states. The model provides analytic expressions for the electric field dependence of the primary charge separation rate constant, prompt and delayed fluorescence, and predicts an effect on the intersystem crossing rate constant. The model provides a mechanism for a reduction of the quantum yield and an explanation for the lack of a concomitant increase in either prompt or delayed fluorescence.

Folates are ubiquitous cofactors that participate in a wide variety of critical biological processes. 5,10-Methenyltetrahydrofolate and its photodegradation product 5,10-methylenetetrahydrofolate are both associated with the light-driven DNA repair protein DNA photolyase and its homologues (e.g., cryptochromes). The excited state electronic properties of these folate molecules have been studied here using Stark spectroscopy and complementary quantum calculations. The tetrahydrofolates have relatively large difference dipole moments (ca. 6-8 Debye) and difference polarizabilities (ca. 100 Å(3)). This extensive excited state charge redistribution appears to be due largely to the pendant p-aminobenzoic acid group, which helps shuttle charge over the entirety of the molecule. Simple calculations based on the experimental difference dipole moments suggest that tetrahydrofolates should have large two photon cross sections sufficient to enable two photon microscopy to selectively detect and follow folate-containing proteins both in vitro and in vivo.

Flavins and flavoproteins have been studied by a plethora of spectroscopic techniques. Beginning with the characterization of DNA photolyases and the discovery of the diversity of roles played by excited-state flavins in photobiology, the characterization of the electronic excited state of flavins has become increasingly important. In this protocol, we provide a guide to using Stark spectroscopy in obtaining the degree of electronic charge redistribution in simple flavins and in flavoproteins. Stark spectroscopy is technically simpler than more common approaches used to explore the structure of the excited state, considerably cheaper to implement, and yet very powerful in its scope. At the end of this guide, we present data taken on non-photobiological flavoproteins, glutathione reductase and lipoamide dehydrogenase, that suggest that Stark spectroscopy is a unique way to elucidate the electrostatic environment that the flavin cofactor experiences bound inside the protein.

Flavins have unique electronic properties that have led to their utilization in biological redox chemistry. Despite this there is relatively little experimental information about such basic electronic properties as dipole moments and polarizabilities for these molecules. We have explored the electronic structure of the ground and first two excited electronic states of an oxidized flavin in nonpolar organic glasses using Stark spectroscopy. The dipole moment change for the S0 → S1 transition is about 3 times smaller than the dipole moment change for the S0 → S2 transition. Dipole moment directions for these excited states have been calculated using the experimental results along with predictions from calculations. The experimental results are consistent with computational predictions of intramolecular charge transfer for the S0 → S2 transition. In addition, we have verified that there is a large change in polarizability for the S2 state vs the S0 and S1 states. Both excited-state polarizability changes sho...

DNA photolyase is a flavoprotein that repairs cyclobutylpyrimidine dimers by ultrafast photoinduced electron transfer. One unusual feature of this enzyme is the configuration of the FAD cofactor, where the isoalloxazine and adenine rings are nearly in vdW contact. We have measured the steady-state and transient absorption spectra and excited-state decay kinetics of oxidized (FAD-containing, folate-depleted) Escherichia coli DNA photolyase with and without dinucleotide and polynucleotide single-stranded thymidine dimer substrates. The steady-state absorption spectrum for the enzyme-polynucleotide substrate complex showed a blue shift, as seen previously by Jorns et al. (1). No shift was observed for the dinucleotide substrate, suggesting that there are significant differences in the binding geometry of dinucleotide versus polynucleotide dimer lesions. Evidence was obtained from transient absorption experiments for a long-lived charge-transfer complex involving the isoalloxazine of the FAD cofactor. No evidence of excited-state quenching was measurable upon binding either substrate. To explain these data, we hypothesize the existence of a large substrate electric field in the cavity containing the FAD cofactor. A calculation of the magnitude and direction of this dipolar electric field is consistent with electrochromic band shifts for both S(0) --> S(1) and S(0) --> S(2) transitions. These observations suggest that the substrate dipolar electric field may be a critical component in its electron-transfer-mediated repair by photolyase and that the unique relative orientation of the isoalloxazine and adenine rings may have resulted from the consequences of the dipolar substrate field.

Flavins and flavoproteins are versatile redox cofactors that can perform both one- and two-electron transfer. Because they are highly colored in all three oxidation states, optical spectroscopy has been exploited for decades to study these redox changes. This review summarizes the application of optical spectroscopies to flavins and flavoproteins since 1990. Special emphasis is placed on new techniques, such as Stark spectroscopy, as well as significant refinements in more well known techniques, such as resonance Raman spectroscopy and ultrafast spectroscopy.

We have measured the excited-state dynamics and spectral evolution of oxidized flavin−adenine dinucleotide (FADox) and flavin mononucleotide (FMNox) in simple solvents with subpicosecond time resolution by one-color and white-light continuum transient absorption spectroscopy. In water, the FADox transient shows significant quenching of the excited state with a lifetime of ∼4 ps while for FMNox this component is of much lower amplitude. However, when caffeine is added to the FMN solution the amplitude of the fast component is recovered. Additionally, the excited-state dynamics of FAD in neat formamide, a solvent that breaks up stacking interactions in FAD, is similar to that obtained for FMN in water. These measurements provide conclusive evidence that the excited-state quenching observed in FAD or flavin−purine complexes occurs in less than 5 ps. The observed spectral evolution shows that internal conversion from S2 to S1 occurs in less than 100 fs.

Stark-effect spectroscopy (electroabsorption) measurements were obtained for oxidized flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in frozen glycerol/H2O glasses and N(3)-methyl-N(10)-isobutyl-7,8-dimethyl-isoalloxazine in frozen n-butanol glasses at fields of up to 5 × 105 V/cm. In all three flavins, the effect of the applied electric field on the low-energy transition (S0 → S1, 450 nm band) is significantly smaller than on the higher energy transition (S0 → S2, 370 nm band). The Stark spectra indicate that the magnitude of the permanent dipole moment in the S1 state, |μ1| is only modestly different from the S0 state, |μ0|, and that there is little change in the mean polarizability for the S0 → S1 transition. The electric field effect on the S0 → S2 transition, however, shows that the magnitude of the dipole moment of the S2 state is ∼60% larger than that of the S1 state and that the change in the mean polarizability is much larger. Concentration studies indicate that the FAD dimer o...

The surface shape of a paraboloidal ree ector was recone gured using electrostatic forces of repulsion. The experiment consisted of a recone gurable electrostatically shaped ree ector and a measurement system. A set of test cases demonstrated pointing and beam-width control of the radiating surface. The surface ree ector angles of rotation µx and µy were plotted vs eight test cases. The resulting sine functions show the near-linear relationship between voltage and pointing, indicating that expected rotations are in agreement with measurements and that a simple interpretation of Coulomb’ s law dominates the behavior of the system. Nomenclature a = vector of undetermined coefe cients C = coefe cient matrix Ex = error function for rotation about the x axis Ey = error function for rotation about the y axis i; j;k = counting indices p = number of sample points in one quadrant Rx;Ry = rotation matrices xn; yn;zn = coordinates in a new reference frame z = vertical position of ith point in the (j;k)th quadrant z = new vertical position of ith point in the (j;k)th quadrant z = vector of measured displacements z (x; y) = function to be curve e tted µx = angle of rotation about the x axis µy = angle of rotation about the y axis

Flavins and flavoproteins have been studied by a plethora of spectroscopic techniques. Beginning with the characterization of DNA photolyases and the discovery of the diversity of roles played by excited-state flavins in photobiology, the characterization of the electronic excited state of flavins has become increasingly important. In this protocol, we provide a guide to using Stark spectroscopy in obtaining the degree of electronic charge redistribution in simple flavins and in flavoproteins. Stark spectroscopy is technically simpler than more common approaches used to explore the structure of the excited state, considerably cheaper to implement, and yet very powerful in its scope. At the end of this guide, we present data taken on non-photobiological flavoproteins, glutathione reductase and lipoamide dehydrogenase, that suggest that Stark spectroscopy is a unique way to elucidate the electrostatic environment that the flavin cofactor experiences bound inside the protein.

Breast-cancer risk factors, mammography history, and breast cancer detection rates were compared for women aged 50–64 years versus women 65 years and older. Mammographic screening examinations were performed on 936 women at least 65 years old and 1,926 women aged 50–64 years at a mobile van and an outpatient clinic of a tertiary referral university hospital. Breast-cancer risk factors and frequency and timing of previous mammograms were assessed on all women. Of the 2,862 women screened, 520 (18.2%) were black. Women aged 65 and older were significantly more likely to be nulliparous, and over 30 years old at first pregnancy than women aged 50–64 years. The proportion of women who reported ever having a mammogram did not differ by age. For women who reported a previous mammogram, the intervals of the previous screening did not differ significantly by age group. Cancer detection rates were higher among women aged 65 and over at both locations. Among age and race groupings, the highest cancer detection rate occurred in black women over 65 years (19.7/1,000). Cancer detection rates were high enough to suggest that screening elderly women is efficacious. Because elderly black women may be less aware of breast-cancer risk factors, relying on self-referral for screening this population may be inappropriate. Barriers to mammographic screening among elderly women warrant further study.

Ultrafast singlet excited state energy transfer occurs from the monomeric chromophores to the primary electron donor or special pair in photosynthetic reaction centers. The mechanism of this proces...

Psoralens are a class of pharmaceutical agents commonly used to treat several cutaneous disorders. When irradiated with a mode-locked titanium: sapphire (Ti:sapphire) laser tuned to 730 nm, an aqueous solution of 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) emits blue light. The emission spectrum is centered at 452 nm and is identical to that obtained by one-photon excitation with UVA excitation, and its magnitude depends quadratically on the intensity of laser excitation. These results suggest that two-photon excitation occurs to a potentially photochemically active state. To estimate the two-photon absorption cross section, it was first necessary to measure the emission quantum yield of HMT using 365 nm excitation at room temperature that resulted in a value of 0.045 +/- 0.007. The two-photon absorption cross section of HMT at 730 nm is therefore estimated to be 20 x 10(-50) cm4 s (20 Göppert-Mayer). The excited-state photophysics and photochemistry of psoralens suggest potential applications to cutaneous phototherapy in diseases such as psoriasis and dystrophic epidermolysis bullosa.

Chromophores containing a donor-π-acceptor (D-π-A) motif have been shown to exhibit many interesting photophysical properties. The lowest electronic transition of a flavin derivative containing this motif, azobenzylflavin (ABFL), has previously been shown to be highly sensitive to solvent environment and hydrogen bonding ligands. To better understand this sensitivity, we have investigated the excited state charge redistribution and dynamics of ABFL in a low-dielectric, non-hydrogen bonding solvent by steady-state Stark and femtosecond optical transient absorption spectroscopies. The Stark measurements reveal the difference dipole moment, Δμ01, between the ground and first excited states to be 22.3 ± 0.9 D. The direction of Δμ01 in the molecular frame was assigned with the aid of TD-DFT and finite field calculations, verifying the hypothesis that electron density moves from the diethylaniline donor to the flavin acceptor in the excited state. The magnitude of the difference dipole moment was used to estimate the hyperpolarizability of ABFL, β0 = 720 × 10(-30) esu. Subsequent excited state decay via charge recombination was shown to take place in a few picoseconds. The data was best fit to a kinetic model composed of a sub-picosecond internal conversion step from S2→S1, followed by a 5 ps decay to the ground state. A competing process involving formation of an additional long-lived state from S1 was also observed. Cyclic voltammetry shows one oxidation and two reduction waves and is completely reversible. This analysis lays the groundwork for developing new flavin dyads with the desired excited electronic state properties for applications such as nonlinear optical devices, molecular electronics applications, or dye-sensitized solar cells.

A new type of adaptable antenna is described that points over a 6-deg range and whose directivity is varied over a 6.5-dB range in experimental tests. The electrostatic antenna concept presented here can be regarded as a compromise between the low-performance mechanical method of adapting the far-field beam pattern and the high-performance but often prohibitively complex method of carrying out the adaptations electronically. The electrostatic antenna is shown to be well suited for space-based applications.

A case of delayed diagnosis of a pancreatic transection after blunt abdominal trauma is presented. The cause of diagnostic delay as well as measures to avoid future errors in diagnosis are outlined.