Multiscale Transient Absorption Study of the Fluorescent Protein Dreiklang and Two Point Variants Provides Insight into Photoswitching and Nonproductive Reaction Pathways.

Dreiklang is a reversibly photoswitchable fluorescent protein used as a probe in advanced fluorescence imaging. It undergoes a unique and still poorly understood photoswitching mechanism based on the reversible addition of a water molecule to the chromophore. We report the first comprehensive study of the dynamics of this reaction by transient absorption spectroscopy from 100 fs to seconds in the original Dreiklang protein and two point variants.

Systematics and Phylogenetic Interrelationships of the Enigmatic Late Jurassic Shark Woodward, 1918 with Comments on the Shark-Ray Sister Group Relationship.

The Late Jurassic elasmobranch is often regarded as a key species to our understanding of crown group elasmobranch interrelationships and the evolutionary history of this group. However, since its first description more than 100 years ago, its phylogenetic position within the Elasmobranchii (sharks and rays) has proven controversial, and a closer relationship between and each of the posited superorders (Batomorphii, Squalomorphii, and Galeomorphii) has been proposed over the time. Here we revise this controversial taxon based on new holomorphic specimens from the Late Jurassic Konservat-Lagerstätte of the Solnhofen Archipelago in Bavaria (Germany) and review its skeletal morphology, systematics, and phylogenetic interrelationships.

Ultrafast dynamics of fully reduced flavin in catalytic structures of thymidylate synthase ThyX.

Thymidylate is a vital DNA precursor synthesized by thymidylate synthases. ThyX is a flavin-dependent thymidylate synthase found in several human pathogens and absent in humans, which makes it a potential target for antimicrobial drugs. This enzyme methylates the 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate (dTMP) using a reduced flavin adenine dinucleotide (FADH) as prosthetic group and (6)-,-methylene-5,6,7,8-tetrahydrofolate (CHTHF) as a methylene donor.

Ultrafast photoreduction dynamics of a new class of CPD photolyases.

NewPHL is a recently discovered subgroup of ancestral DNA photolyases. Its domain architecture displays pronounced differences from that of canonical photolyases, in particular at the level of the characteristic electron transfer chain, which is limited to merely two tryptophans, instead of the "classical" three or four. Using transient absorption spectroscopy, we show that the dynamics of photoreduction of the oxidized FAD cofactor in the NewPHL begins similarly as that in canonical photolyases, i.

Ultrafast Oxidation of a Tyrosine by Proton-Coupled Electron Transfer Promotes Light Activation of an Animal-like Cryptochrome.

The animal-like cryptochrome of (aCRY) is a recently discovered photoreceptor that controls the transcriptional profile and sexual life cycle of this alga by both blue and red light. aCRY has the uncommon feature of efficient formation and longevity of the semireduced neutral form of its FAD cofactor upon blue light illumination. Tyrosine Y plays a crucial role by elongating , as fourth member, the electron transfer (ET) chain found in most other cryptochromes and DNA photolyases, which comprises a conserved tryptophan triad.

Ultrafast photoinduced flavin dynamics in the unusual active site of the tRNA methyltransferase TrmFO.

Flavoproteins often stabilize their flavin coenzyme by stacking interactions involving the isoalloxazine moiety of the flavin and an aromatic residue from the apoprotein. The bacterial FAD and folate-dependent tRNA methyltransferase TrmFO has the unique property of stabilizing its FAD coenzyme by an unusual H-bond-assisted π-π stacking interaction, involving a conserved tyrosine (Y346 in Bacillus subtilis TrmFO, BsTrmFO), the isoalloxazine of FAD and the backbone of a catalytic cysteine (C53). Here, the interaction between FAD and Y346 has been investigated by measuring the photoinduced flavin dynamics of BsTrmFO in the wild-type (WT) protein, C53A and several Y346 mutants by ultrafast transient absorption spectroscopy.

Delocalized hole transport coupled to sub-ns tryptophanyl deprotonation promotes photoreduction of class II photolyases.

Class II photolyases utilize for the photoreduction of their flavin cofactor (FAD) a completely different tryptophan triad than most other photolyases and cryptochromes. To counter sped-up back electron transfer, they evolved an unusually fast deprotonation of the distal tryptophanyl radical cation (WH˙+) that is produced after excitation of the flavin. We studied the primary aspects of oxidized FAD photoreduction by ultrafast transient absorption spectroscopy, using the class II photolyase from Methanosarcina mazei.

Ultrafast flavin photoreduction in an oxidized animal (6-4) photolyase through an unconventional tryptophan tetrad.

Photolyases are flavoenzymes repairing UV-induced lesions in DNA, which may be activated by a photoreduction of their FAD cofactor. In most photolyases, this photoreduction proceeds by electron transfer along a chain of three tryptophan (Trp) residues, connecting the flavin to the protein surface. Much less studied, animal (6-4) photolyases (repairing pyrimidine-pyrimidone (6-4) photoproducts) are particularly interesting as they were recently shown to have a longer electron transfer chain, counting four Trp residues.

Photoinduced Chromophore Hydration in the Fluorescent Protein Dreiklang Is Triggered by Ultrafast Excited-State Proton Transfer Coupled to a Low-Frequency Vibration.

Because of growing applications in advanced fluorescence imaging, the mechanisms and dynamics of photoinduced reactions in reversibly photoswitchable fluorescent proteins are currently attracting much interest. We report the first time-resolved study of the photoswitching of Dreiklang, so far the only fluorescent protein to undergo reversible photoinduced chromophore hydration. Using broadband femtosecond transient absorption spectroscopy, we show that the reaction is triggered by an ultrafast deprotonation of the chromophore phenol group in the excited state in 100 fs.

Ultrafast Dynamics of a Green Fluorescent Protein Chromophore Analogue: Competition between Excited-State Proton Transfer and Torsional Relaxation.

The competition between excited-state proton transfer (ESPT) and torsion plays a central role in the photophysics of fluorescent proteins of the green fluorescent protein (GFP) family and their chromophores. Here, it was investigated in a single GFP chromophore analogue bearing o-hydroxy and p-diethylamino substituents, OHIM. The light-induced dynamics of OHIM was studied by femtosecond transient absorption spectroscopy, at different pH.

Photo-induced cation translocation in a molecular shuttle based on a calix[4]-biscrown including DCM and DMABN chromophores.

We present a new molecular shuttle, consisting of a calixarene core attached to two different photoactive centers, DCM and DMABN. We show that a K(+) ion bound to the DCM-grafted crown is translocated towards the other site of the molecule upon photoexcitation, but not released to the bulk.

Real-time monitoring of chromophore isomerization and deprotonation during the photoactivation of the fluorescent protein Dronpa.

Dronpa is a photochromic green fluorescent protein (GFP) homologue used as a probe in super-resolution microscopy. It is known that the photochromic reaction involves cis/trans isomerization of the chromophore and protonation/deprotonation of its phenol group, but the sequence in time of the two steps and their characteristic time scales are still the subject of much debate. We report here a comprehensive UV-visible transient absorption spectroscopy study of the photoactivation mechanism of Dronpa, covering all relevant time scales from ∼100 fs to milliseconds.

New insights into the ultrafast photophysics of oxidized and reduced FAD in solution.

The ultrafast photophysics of oxidized and reduced flavin adenine dinucleotide (FAD) in aqueous solution was studied by broadband UV-vis femtosecond transient absorption spectroscopy. We observed that oxidized FAD (FAD(ox)) in solution readily aggregates at submillimolar concentration. Upon excitation of FAD(ox), three excited-state lifetimes were found and assigned to three different species: the closed (stacked) conformation of the monomer (∼5.

Primary photodynamics of a biomimetic model of photoactive yellow protein (PYP).

The present work aims at characterizing the photophysical behavior of a first biomimetic cyclodextrin model (CD-PYP1) of the photoactive site of photoactive yellow protein (PYP). The hydrophobic cyclodextrin cavity in which the chromophore self-includes, mimics its local environment within the protein. The photoinduced behavior of deprotonated CD-PYP1 (dp-CD-PYP1) has been probed by femtosecond transient-absorption spectroscopy and compared to those of the free deprotonated chromophore (pCT(-)) and of wild-type PYP.

Photoinduced cation translocation in a calix[4]biscrown: towards a new type of light-driven molecular shuttle.

The photophysics of a ditopic receptor of potassium ion consisting of a 1,3-alternate calix[4]biscrown with a merocyanine dye (DCM) inserted into each crown is reported. Thanks to the large difference between the binding affinity for one and two potassium ions, one can find relative total concentrations of ligand and potassium ion at which the 1:1 complex is most predominant with respect to the free ligand and the 2:1 complex whose amounts are a few percents. Investigation of the 1:1 complex by femtosecond transient absorption spectroscopy provides evidence for the ultrafast movement of a potassium ion through the calix[4]arene tube upon excitation at 400 nm of the dye.

Ultrafast delocalization of cationic states in poly(N-vinylcarbazole) solid leading to carrier photogeneration.

Femtosecond measurements of the transient dichroism and near-IR time-resolved spectra revealed the ultrafast delocalization of the cationic state in poly(N-vinylcarbazole), leading to carrier photogeneration.

Spectro-temporal characterization of the photoactivation mechanism of two new oxidized cryptochrome/photolyase photoreceptors.

The photoactivation dynamics of two new flavoproteins (OtCPF1 and OtCPF2) of the cryptochrome photolyase family (CPF), belonging to the green alga Ostreococcus tauri , was studied by broadband UV-vis femtosecond absorption spectroscopy. Upon excitation of the protein chromophoric cofactor, flavin adenine dinucleotide in its oxidized form (FAD(ox)), we observed in both cases the ultrafast photoreduction of FAD(ox): in 390 fs for OtCPF1 and 590 fs for OtCPF2. Although such ultrafast electron transfer has already been reported for other flavoproteins and CPF members, the present result is the first demonstration with full spectral characterization of the mechanism.

Femtosecond to subnanosecond multistep calcium photoejection from a crown ether-linked merocyanine.

Photoinduced calcium release from the crown ether-linked merocyanine DCM-crown is reexamined by femtosecond transient absorption spectroscopy with sub-100 fs time resolution. Photodisruption of the bond linking the cation to the nitrogen atom shared by the crown and the chromophore is found to take place in 130 fs. Confirming our previous reports, the photoinduced intraligand charge transfer is observed in the picosecond regime but kinetics involving three-components (1 ps, 8 ps and 77 ps), together with a 56 ps stimulated-emission time-resolved red shift, are found in the present study.

Primary photoprocesses involved in the sensory protein for the photophobic response of Blepharisma japonicum.

We present new femtosecond transient-absorption and picosecond fluorescence experiments performed on OBIP, the oxyblepharismin-binding protein believed to trigger the photophobic response of the ciliate Blepharisma japonicum. The formerly identified heterogeneity of the sample is confirmed and rationalized in terms of two independent populations, called rOBIP and nrOBIP. The rOBIP population undergoes a fast photocycle restoring the initial ground state in less than 500 ps.

Steady-state and femtosecond photoinduced processes of blepharismins bound to alpha-crystallin.

The interaction of blepharismin (BP) and oxyblepharismin (OxyBP) with bovine alpha-crystallin (BAC) has been studied both by steady-state and femtosecond spectroscopy, with the aim of assessing the possible phototoxicity of these compounds toward the eye tissues. We showed that these pigments form with BAC potentially harmful ground-state complexes, the dissociation constants of which have been estimated to be 6 +/- 2 micromol L(-1) for OxyBP and 9 +/- 4 micromol L(-1) for BP. Irradiation with steady-state visible light of solutions of blepharismins in the presence of BAC proved to induce a quenching of both the pigment and the intrinsic protein fluorescences.