Comparing ultrafast excited state quenching of flavin 1,N6-ethenoadenine dinucleotide and flavin adenine dinucleotide by optical spectroscopy and DFT calculations.

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∙+ and Fl∙- radicals is elusive. Here we use the FAD analog Flavin 1,N6-Ethenoadenine Dinucleotide (εFAD) to study how different photochemical outcomes depend on the identity of the Ade moiety in stacked FAD and its analog εFAD. We have used UV-Vis transient absorption spectroscopy complemented by TD-DFT calculations to investigate the excited state evolution of the flavins. In FAD*, no radicals were observed, suggesting that FAD* does not undergo PET. εFAD* kinetics showed a broad absorption band that suggests a charge transfer state exists upon photoexcitation with evidence for radical pair formation. Surprisingly, significant triplet flavin was produced from εFAD* We hypothesize that the dipolar (ε)Ade moieties differentially modulate the singlet-triplet energy gap, resulting in different intersystem crossing rates. The additional electron density on the etheno group of εFAD supplies better orbital overlap with the flavin S1 state, accelerating charge transfer in that molecule.

Rosa26-GFP direct repeat (RaDR-GFP) mice reveal tissue- and age-dependence of homologous recombination in mammals in vivo.

Homologous recombination (HR) is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP) mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals.

Saturation Dynamics Measures Absolute Cross Section and Generates Contrast within a Neuron.

The intensity required to optically saturate a chromophore is a molecular property that is determined by its absorption cross section (σ) and the excited state lifetime. We present an analytical description of such a system and show that fluorescence around the onset of saturation is characterized by product of absorption cross section and lifetime. Using this approach we formulate a generalized method for measuring the multiphoton cross section of fluorophores and use it to obtain the absolute three-photon cross-section spectra of tryptophan. We find that the tryptophan three-photon cross section ranges from 0.28 S.I. units (m(6)s(2)photon(-2)) at 870 nm to 20 S.I. units at 740 nm. Further, we show that the product of molecular rate of excitation and de-excitation, denoted as ß, serves as a vital contrasting agent for imaging local environment. Our contrast parameter, ß, is related to fraction of the population present in the excited state and is independent of the fluorophore concentration. We show that ß-imaging can be carried out in a regular two-photon microscope setup through a series of intensity scans. Using enhanced green fluorescent protein (EGFP) fluorescence from the brain slices of Thy-1 EGFP transgenic mice, we show that there is an inherent, concentration independent, variation in contrast across the soma and the dendrite.

A soft X-ray spectroscopic perspective of electron localization and transport in tungsten doped bismuth vanadate single crystals.

Doped BiVO4 is a promising photoelectrochemical water splitting anode, whose activity is hampered by poor charge transport. Here we use a set of X-ray spectroscopic methods to probe the origin and nature of localized electron states in W:BiVO4. Furthermore, using the polarized nature of the X-rays, we probe variations in the electronic structure along the crystal axes. In this manner, we reveal aspects of the electronic structure related to electron localization and observations consistent with conductivity anisotropy between the ab-plane and c-axis. We verify that tungsten substitutes as W6+ for V5+ in BiVO4. This is shown to result in the presence of inter-band gap states related to electrons at V4+ sites of e symmetry. The energetic position of the states in the band gap suggest that they are highly localized and may act as recombination centres. Polarization dependent X-ray absorption spectra reveal anisotropy in the electronic structure between the ab-plane and c-axis. Results show the superior hybridization between V 3d and O 2p states, higher V wavefunction overlap and broader conduction bands in the ab-plane than in the c-axis. These insights into the electronic structure are discussed in the context of existing experimental and theoretical reports regarding charge transport in BiVO4.

The targeting of the oxysterol-binding protein ORP3a to the endoplasmic reticulum relies on the plant VAP33 homolog PVA12.

In plants, sterols play fundamental roles as membrane constituents in the biosynthesis of steroid hormones, and act as precursors for cell wall deposition. Sterols are synthesized in the endoplasmic reticulum (ER), but mainly accumulate in the plasma membrane. How sterols are trafficked in plant cells is largely unknown. In non-plant systems, oxysterol-binding proteins have been involved in sterol trafficking and homeostasis. There are at least twelve homologs of oxysterol-binding proteins in the Arabidopsis genome, but the biology of these proteins remains for the most part obscure. Here, we report our analysis of the targeting requirements and the sterol-binding properties of a small Arabidopsis oxysterol-binding protein, ORP3a. We have determined that ORP3a is a bona fide sterol-binding protein with sitosterol-binding properties. Live-cell imaging analyses revealed that ORP3a is localized at the ER, and that binding to this organelle depends on a direct interaction with PVA12, a member of the largely uncharacterized VAP33 family of plant proteins. Molecular modeling analyses and site-directed mutagenesis led to the identification of a novel protein domain that is responsible for the PVA12-ORP3a interaction. Disruption of the integrity of this domain caused redistribution of ORP3a to the Golgi apparatus, suggesting that ORP3a may cycle between the ER and the Golgi. These results represent new insights into the biology of sterol-binding proteins in plant cells, and elucidate a hitherto unknown relationship between members of oxysterol-binding protein and VAP33 families of plant proteins in the early plant secretory pathway.

Unfolded-state dynamics and structure of protein L characterized by simulation and experiment.

While several experimental techniques now exist for characterizing protein unfolded states, all-atom simulation of unfolded states has been challenging due to the long time scales and conformational sampling required. We address this problem by using a combination of accelerated calculations on graphics processor units and distributed computing to simulate tens of thousands of molecular dynamics trajectories each up to approximately 10 mus (for a total aggregate simulation time of 127 ms). We used this approach in conjunction with Trp-Cys contact quenching experiments to characterize the unfolded structure and dynamics of protein L. We employed a polymer theory method to make quantitative comparisons between high-temperature simulated and chemically denatured experimental ensembles and find that reaction-limited quenching rates calculated from simulation agree remarkably well with experiment. In both experiment and simulation, we find that unfolded-state intramolecular diffusion rates are very slow compared to highly denatured chains and that a single-residue mutation can significantly alter unfolded-state dynamics and structure. This work suggests a view of the unfolded state in which surprisingly low diffusion rates could limit folding and opens the door for all-atom molecular simulation to be a useful predictive tool for characterizing protein unfolded states along with experiments that directly measure intramolecular diffusion.

The intrinsic stiffness of polyglutamine peptides.

We have used the method of Trp/Cys contact quenching to measure the rate of contact formation in polyglutamine and find it to be a very stiff peptide. Separation of observed rates into reaction-limited and diffusion-limited rates show that the reaction-limited rates increase (rather than decrease) slightly with length between 4 and 16 amino acids. Using Szabo, Schulten, and Schulten theory, we have modeled the results with a wormlike chain with excluded volume and find the persistence length to be about 13.0 A, much longer than has been observed for other random peptides and unfolded proteins. The preferred extended conformation of polyglutamine could account for a propensity for expanded glutamine stretches to unfold the Huntington's protein and the high propensity to aggregate from a disordered monomer.

An Ethenoadenine FAD Analog Accelerates UV Dimer Repair by DNA Photolyase.

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 (kET ) 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.

Ruggedness in the folding landscape of protein L.

By exploring the folding pathways of the B1 domain of protein L with a series of equilibrium and rapid kinetic experiments, we have found its unfolded state to be more complex than suggested by two-state folding models. Using an ultrarapid mixer to initiate protein folding within approximately 2-4 microseconds, we observe folding kinetics by intrinsic tryptophan fluorescence and fluorescence resonance energy transfer. We detect at least two processes faster than 100 mus that would be hidden within the burst phase of a stopped-flow instrument measuring tryptophan fluorescence. Previously reported measurements of slow intramolecular diffusion are commensurate with the slower of the two observed fast phases. These results suggest that a multidimensional energy landscape is necessary to describe the folding of protein L, and that the dynamics of the unfolded state is dominated by multiple small energy barriers.

Dynamic similarity of the unfolded states of proteins L and G.

The formation of specific intramolecular contacts has been studied under a range of denaturing conditions in single domains of the immunoglobulin-binding proteins L and G. Although they share no significant sequence similarity and have dissimilar folding pathways, the two domains have a similar native fold. Our measurements show that the rates of forming corresponding contacts in the unfolded states of both proteins are remarkably similar and even exhibit similar dependence on denaturant concentration. The unfolded proteins were modeled using Szabo, Schulten, and Schulten (SSS) theory as wormlike chains with excluded volume; when combined with our experimental data, the SSS analysis suggests that the unfolded state becomes uniformly more compact and less diffusive (i.e., rearranges more slowly) with decreasing denaturant concentrations.