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Phytochrome proteins regulate many photoresponses of plants and microorganisms. Light absorption causes isomerization of the biliverdin chromophore, which triggers a series of structural changes to activate the signaling domains of the protein. However, the structural changes are elusive, and therefore the molecular mechanism of signal transduction remains poorly understood. Here, we apply two-color step-scan infrared spectroscopy to the bacteriophytochrome from Deinococcus radiodurans. We show by recordings in H2O and D2O that the hydrogen bonds to the biliverdin D-ring carbonyl become disordered in the first intermediate (Lumi-R) forming a dynamic microenvironment, then completely detach in the second intermediate (Meta-R), and finally reform in the signaling state (Pfr). The spectra reveal via isotope labeling that the refolding of the conserved “PHY-tongue” region occurs with the last transition between Meta-R and Pfr. Additional changes in the protein backbone are detected already within microseconds in Lumi-R. Aided by molecular dynamics simulations, we find that a strictly conserved salt bridge between an arginine of the PHY tongue and an aspartate of the chromophore binding domains is broken in Lumi-R and the arginine is recruited to the D-ring C═O. This rationalizes how isomerization of the chromophore is linked to the global structural rearrangement in the sensory receptor. Our findings advance the structural understanding of phytochrome photoactivation. peerReviewed

Photosynthetic reaction centers convert sunlight into a transmembrane electrochemical potential difference, providing chemical energy to almost all life on earth. Light energy is efficiently transferred through chromophore cofactors to the sites, where charge separation occurs. We applied two-dimensional electronic spectroscopy to assess the role of coherences in the photoresponse of the bacterial reaction center of Rhodobacter sphaeroides. By controlling the polarization of the laser beams, we were able to assign unambiguously the oscillatory dynamics to electronic (intermolecular) coherences. The data show that these coherences are sustained for more than 1 ps, indicating that the protein coherently retains some excitation energy on this time scale. Our finding provides a mechanism for effective delocalization of the excitations on the picosecond time scale by electronic coherence, setting the stage for efficient charge separation.

Bimolecular interactions between excitations in conjugated polymer thin films are important because they influence the efficiency of many optoelectronic devices that require high excitation densities. Using time-resolved optical spectroscopy, we measure the bimolecular interactions of charges, singlet excitons, and triplet excitons in intimately mixed polyfluorene blends with band-edge offsets optimized for photoinduced electron transfer. Bimolecular charge recombination and triplet-triplet annihilation are negligible, but exciton-charge interactions are efficient. The annihilation of singlet excitons by charges occurs on picosecond time-scales and reaches a rate equivalent to that of charge transfer. Triplet exciton annihilation by charges occurs on nanosecond time-scales. The surprising absence of nongeminate charge recombination is shown to be due to the limited mobility of charge carriers at the heterojunction. Therefore, extremely high densities of charge pairs can be maintained in the blend. The absence of triplet-triplet annihilation is a consequence of restricted triplet diffusion in the blend morphology. We suggest that the rate and nature of bimolecular interactions are determined by the stochastic excitation distribution in the polymer blend and the limited connectivity between the polymer domains. A model based on these assumptions quantitatively explains the effects. Our findings provide a comprehensive framework for understanding bimolecular recombination and annihilation processes in nanostructured materials.

Vibrational spectra contain unique information on protein structure and dynamics. However, this information is often obscured by spectral congestion, and site-selective information is not available. In principle, sites of interest can be spectrally identified by isotope shifts, but site-specific isotope labeling of proteins is today possible only for favorable amino acids or with prohibitively low yields. Here we present an efficient cell-free expression system for the site-specific incorporation of any isotope-labeled amino acid into proteins. We synthesized 1.6 mg of green fluorescent protein with an isotope-labeled tyrosine from 100 mL of cell-free reaction extract. We unambiguously identified spectral features of the tyrosine in the fingerprint region of the time-resolved infrared absorption spectra. Kinetic analysis confirmed the existence of an intermediate state between photoexcitation and proton transfer that lives for 3 ps. Our method lifts vibrational spectroscopy of proteins to a higher level of structural specificity.

A detailed charge recombination mechanism is presented for organic photovoltaic devices with a high open-circuit voltage. In a binary blend comprised of polyfluorene copolymers, the performance-limiting process is found to be the efficient recombination of tightly bound charge pairs into neutral triplet excitons. We arrive at this conclusion using optical transient absorption (TA) spectroscopy with visible and IR probes and over seven decades of time resolution. By resolving the polarization of the TA signal, we track the movement of polaronic states generated at the heterojunction not only in time but also in space. It is found that the photogenerated charge pairs are remarkably immobile at the heterojunction during their lifetime. The charge pairs are shown to be subject to efficient intersystem crossing and terminally recombine into F8BT triplet excitons within approximately 40 ns. Long-range charge separation competes rather unfavorably with intersystem crossing--75% of all charge pairs decay into triplet excitons. Triplet exciton states are thermodynamically accessible in polymer solar cells with high open circuit voltage, and we therefore suggest this loss mechanism to be general. We discuss guidelines for the design of the next generation of organic photovoltaic materials where separating the metastable interfacial charge pairs within approximately 40 ns is paramount.

The conjugation of nanoparticles (NPs) typically yields supramolecular materials which are fairly rigid, and the electronic coupling between the NP and other structural units of these compounds is fixed by covalent bonds. Here, we report on a novel bichromophor system constructed from a quantum dot tethered to a semiconducting polymer, which demonstrates the possibility of the dynamic interunit coupling in the NP supramolecules. The NP bichromophoric system was made on the basis of the layer-by-layer assembled (LBL) films of an anionic polyelectrolyte with poly(p-phenylene ethynylene) backbone, aPPE, and poly(allylamine hydrochloride) PAH polycation. To conjugate CdTe NPs to the (aPPE/PAH)(m) LBL film, we took advantage of the reactive groups of NP stabilizer, that is, -COOH, and the aminogroups on PAH. Tethering of CdTe was accomplished by using poly(ethyleneglycole), PEG, chains with two reactive terminals such as t-BOC-NH-PEG-COO-NHS. The evidence for successful conjugation of NPs to the LBL films can be seen both in AFM images and in optical data. The latter also indicate that the light quanta emitted by the NPs originate from the light absorption of the polymer film, which proves the presence of the aPPE--NP energy-transfer process. The average separation distance between the NPs tethered to the LBL films can be changed by altering the dielectric properties of the solvent affecting PEG tether coiling (water/alcohol mixture). The reduced emission intensity of aPPE was found to follow the extension of the PEG tether. The quenching of aPPE is reversible when the original composition of the solvent mixture is restored. Thus, CdTe-PEG-aPPE is an example of an organized NP system with tunable optical coupling. Variable electronic coupling offers a convenient structural platform for new nanotechnological devices for which spatial control translates into a higher level of sophistication. PEG molecules afford a wide variety of polymer chain configurations with different reactive terminals, which makes possible the preparation of diverse NP superstructures.