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Detecting microsecond structural perturbations in biomolecules has wide relevance in biology, chemistry and medicine. Here we show how MHz repetition rates at X-ray free-electron lasers can be used to produce microsecond time-series of protein scattering with exceptionally low noise levels of 0.001%. We demonstrate the approach by examining Jɑ helix unfolding of a light-oxygen-voltage photosensory domain. This time-resolved acquisition strategy is easy to implement and widely applicable for direct observation of structural dynamics of many biochemical processes.

Charge-transfer reactions in proteins are important for life, such as in photolyases which repair DNA, but the role of structural dynamics remains unclear. Here, using femtosecond X-ray crystallography, we report the structural changes that take place while electrons transfer along a chain of four conserved tryptophans in the Drosophila melanogaster(6-4) photolyase. At femto- and picosecond delays, photoreduction of the flavin by the first tryptophan causes directed structural responses at a key asparagine, at a conserved salt bridge, and by rearrangements of nearby water molecules. We detect charge-induced structural changes close to the second tryptophan from 1 ps to 20 ps, identifying a nearby methionine as an active participant in the redox chain, and from 20 ps around the fourth tryptophan. The photolyase undergoes highly directed and carefully timed adaptations of its structure. This questions the validity of the linear solvent response approximation in Marcus theory and indicates that evolution has optimized fast protein fluctuations for optimal charge transfer.

Resolving the structural dynamics of bond breaking, bond formation, and solvation is required for a deeper understanding of solution-phase chemical reactions. In this work, we investigate the photodissociation of triiodide in four solvents using femtosecond time-resolved X-ray solution scattering following 400 nm photoexcitation. Structural analysis of the scattering data resolves the solvent-dependent structural evolution during the bond cleavage, internal rearrangements, solvent-cage escape, and bond reformation in real time. The nature and structure of the reaction intermediates during the recombination are determined, elucidating the full mechanism of photodissociation and recombination on ultrafast time scales. We resolve the structure of the precursor state for recombination as a geminate pair. Further, we determine the size of the solvent cages from the refined structures of the radical pair. The observed structural dynamics present a comprehensive picture of the solvent influence on structure and dynamics of dissociation reactions.

Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography1using an X-ray free-electron laser2to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridison a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.

Phytochromes sense red/far-red light and control many biological processes in plants, fungi, and bacteria. Although the crystal structures of dark- and light-adapted states have been determined, the molecular mechanisms underlying photoactivation remain elusive. Here, we demonstrate that the conserved tongue region of the PHY domain of a 57-kDa photosensory module of Deinococcus radioduransphytochrome changes from a structurally heterogeneous dark state to an ordered, light-activated state. The results were obtained in solution by utilizing a laser-triggered activation approach detected on the atomic level with high-resolution protein NMR spectroscopy. The data suggest that photosignaling of phytochromes relies on careful modulation of structural heterogeneity of the PHY tongue.

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.

Time-resolved X-ray solution scattering is an increasingly popular method to measure conformational changes in proteins. Extracting structural information from the resulting difference X-ray scattering data is a daunting task. We present a method in which the limited but precious information encoded in such scattering curves is combined with the chemical knowledge of molecular force fields. The molecule of interest is then refined toward experimental data using molecular dynamics simulation. Therefore, the energy landscape is biased toward conformations that agree with experimental data. We describe and verify the method, and we provide an implementation in GROMACS.

Time-resolved wide-angle x-ray scattering (TR-WAXS) is an emerging biophysical method which probes protein conformational changes with time. Here we present a comparative TR-WAXS study of native green-absorbing proteorhodopsin (pR) from SAR86 and a halogenated derivative for which the retinal chromophore has been replaced with 13-desmethyl-13-iodoretinal (13-I-pR). Transient absorption spectroscopy differences show that the 13-I-pR photocycle is both accelerated and displays more complex kinetics than native pR. TR-WAXS difference data also reveal that protein structural changes rise and decay an order-of-magnitude more rapidly for 13-I-pR than native pR. Despite these differences, the amplitude and nature of the observed helical motions are not significantly affected by the substitution of the retinal's C-20 methyl group with an iodine atom. Molecular dynamics simulations indicate that a significant increase in free energy is associated with the 13-cisconformation of 13-I-pR, consistent with our observation that the transient 13-I-pR conformational state is reached more rapidly. We conclude that although the conformational trajectory is accelerated, the major transient conformation of pR is unaffected by the substitution of an iodinated retinal chromophore.

Exciton migration!Spectroscopic analyses and extensive molecular dynamics studies revealed a well‐defined 41helix in which the perylene molecules (see figure) form four “helter‐skelter‐like” overlapping pathways along which excitons and electrons can rapidly migrate.

Through controlled annealing of intimately mixed blends of the polyfluorene copolymers poly(9,9‘-dioctylfluorene-co-bis(N,N‘-(4,butylphenyl))bis(N,N‘-phenyl-1,4-phenylene)diamine) (PFB) and poly(9,9‘-dioctylfluorene-co-benzothiadiazole) (F8BT) we observe the change in charge generation dynamics and photovoltaic performance as the length of nanoscale phase separation is varied from 5 nm or less to greater than 40 nm. We find that device efficiency is optimized for a phase separation of ∼20 nm, significantly larger than the exciton diffusion length of ∼5−10 nm. Femtosecond time-resolved transient absorption measurements confirm that the charge generation time is longer and charge generation efficiency is lower in films with a more evolved morphology. Photoluminescence quantum efficiency is also observed to monotonically increase with annealing temperature consistent with a decrease in exciton dissociation resulting from a coarsening of phases. Using a Monte Carlo model of exciton diffusion and dissociation in computer-simulated structures, we infer that the domains have purity of >95% and find good agreement between the observed photoluminescence quenching and measured domain sizes. Charge transport studies of single-carrier devices show that charge transport through the blend does not significantly improve as device performance improves, and photocurrent is observed to scale linearly with light intensity independent of blend morphology and device geometry. We conclude that the recombination of geminate charge pairs is limiting device performance, with the optimum phase separation of 20 nm balancing the efficiency of charge generation and charge separation.

We present a simulation of exciton dynamics in supramolecular assemblies of an oligo-p-phenylenevinylene derivative monofunctionalised with a quadruple hydrogen-bonding group (MOPV). MOPV molecules form helical stacks in dodecane solution through solvophobic and − interactions with thermotropic reversibility. We apply a model of incoherent excitation hopping using a Monte Carlo scheme to extract microscopic physical quantities relevant to energy diffusion and bimolecular annihilation processes within isolated nanostructures. We compare the simulation to ultrafast spectroscopic data, namely photoinduced absorption transients at various excitation fluences, their polarization anisotropy, and the dynamic photoluminescence red-shift. We observe that energy diffusion and bimolecular annihilation processes can be described with the same microscopic model based on a Förster-like model that takes into account the spatial extent of the excited state; these two processes are interconnected via the same underlying physics. We extract a high diffusion coefficient (∼0.08 cm2s-1) over the first few picoseconds following excitation, which plays an important role in dictating the bimolecular annihilation dynamics.

Structural analysis with TR-WAXS reveals a light-activated conformational change in rhodopsin in native membranes.

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