Cryo-EM structures of a bathy phytochrome histidine kinase reveal a unique light-dependent activation mechanism.

Phytochromes are photoreceptor proteins in plants, fungi, and bacteria. They can adopt two photochromic states with differential biochemical responses. The structural changes transducing the signal from the chromophore to the biochemical output modules are poorly understood due to challenges in capturing structures of the dynamic, full-length protein.

Directed ultrafast conformational changes accompany electron transfer in a photolyase as resolved by serial crystallography.

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.

3D-printed sheet jet for stable megahertz liquid sample delivery at X-ray free-electron lasers.

X-ray free-electron lasers (XFELs) can probe chemical and biological reactions as they unfold with unprecedented spatial and temporal resolution. A principal challenge in this pursuit involves the delivery of samples to the X-ray interaction point in such a way that produces data of the highest possible quality and with maximal efficiency. This is hampered by intrinsic constraints posed by the light source and operation within a beamline environment.

Solvent-Dependent Structural Dynamics in the Ultrafast Photodissociation Reaction of Triiodide Observed with Time-Resolved X-ray Solution Scattering.

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.

Ground-state heterogeneity and vibrational energy redistribution in bacterial phytochrome observed with femtosecond 2D IR spectroscopy.

Phytochromes belong to a group of photoreceptor proteins containing a covalently bound biliverdin chromophore that inter-converts between two isomeric forms upon photoexcitation. The existence and stability of the photocycle products are largely determined by the protein sequence and the presence of conserved hydrogen-bonding interactions in the vicinity of the chromophore. The vibrational signatures of biliverdin, however, are often weak and obscured under more intense protein bands, limiting spectroscopic studies of its non-transient signals.

Structural mechanism of signal transduction in a phytochrome histidine kinase.

Phytochrome proteins detect red/far-red light to guide the growth, motion, development and reproduction in plants, fungi, and bacteria. Bacterial phytochromes commonly function as an entrance signal in two-component sensory systems. Despite the availability of three-dimensional structures of phytochromes and other two-component proteins, the conformational changes, which lead to activation of the protein, are not understood.

Protein motions visualized by femtosecond time-resolved crystallography: The case of photosensory vs photosynthetic proteins.

Proteins are dynamic objects and undergo conformational changes when functioning. These changes range from interconversion between states in equilibrium to ultrafast and coherent structural motions within one perturbed state. Time-resolved serial femtosecond crystallography at free-electron X-ray lasers can unravel structural changes with atomic resolution and down to femtosecond time scales.

Structural basis of the radical pair state in photolyases and cryptochromes.

We present the structure of a photoactivated animal (6-4) photolyase in its radical pair state, captured by serial crystallography. We observe how a conserved asparigine moves towards the semiquinone FAD chromophore and stabilizes it by hydrogen bonding. Several amino acids around the final tryptophan radical rearrange, opening it up to the solvent.

The three-dimensional structure of Drosophila melanogaster (6-4) photolyase at room temperature.

(6-4) photolyases are flavoproteins that belong to the photolyase/cryptochrome family. Their function is to repair DNA lesions using visible light. Here, crystal structures of Drosophila melanogaster (6-4) photolyase [Dm(6-4)photolyase] at room and cryogenic temperatures are reported.

Comparative analysis of two paradigm bacteriophytochromes reveals opposite functionalities in two-component signaling.

Bacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome from Deinococcus radiodurans (DrBphP) lacks detectable kinase activity.

Cryo-Electron Microscopy of Phytochrome A in Its Pr State Reveals Head-to-Head Homodimeric Architecture.

Phytochrome photoreceptors regulate vital adaptations of plant development, growth, and physiology depending on the ratio of red and far-red light. The light-triggered / isomerization of a covalently bound bilin chromophore underlies phytochrome photoconversion between the red-absorbing Pr and far-red-absorbing Pfr states. Compared to bacterial phytochromes, the molecular mechanisms of signal propagation to the C-terminal module and its regulation are little understood in plant phytochromes, not least owing to a dearth of structural information.

High-resolution crystal structures of transient intermediates in the phytochrome photocycle.

Phytochromes are red/far-red light photoreceptors in bacteria to plants, which elicit a variety of important physiological responses. They display a reversible photocycle between the resting Pr state and the light-activated Pfr state. Light signals are transduced as structural change through the entire protein to modulate its activity.

Dynamic band-shift signal in two-dimensional electronic spectroscopy: A case of bacterial reaction center.

Optical nonlinear spectroscopies carry a high amount of information about the systems under investigation; however, as they report polarization signals, the resulting spectra are often congested and difficult to interpret. To recover the landscape of energy states and physical processes such as energy and electron transfer, a clear interpretation of the nonlinear signals is prerequisite. Here, we focus on the interpretation of the electrochromic band-shift signal, which is generated when an internal electric field is established in the system following optical excitation.

Observing the Structural Evolution in the Photodissociation of Diiodomethane with Femtosecond Solution X-Ray Scattering.

Resolving the structural dynamics of the initial steps of chemical reactions is challenging. We report the femtosecond time-resolved wide-angle x-ray scattering of the photodissociation of diiodomethane in cyclohexane. The data reveal with structural detail how the molecule dissociates into radicals, how the radicals collide with the solvent, and how they form the photoisomer.

Ultrafast structural changes within a photosynthetic reaction centre.

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 crystallography using an X-ray free-electron laser to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on 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.

Resolution enhancement in NMR spectra by deconvolution with compressed sensing reconstruction.

NMR spectroscopy is one of the basic tools for molecular structure elucidation. Unfortunately, the resolution of the spectra is often limited by inter-nuclear couplings. The existing workarounds often alleviate the problem by trading it for another deficiency, such as spectral artefacts or difficult sample preparation and, thus, are rarely used.

Tips and turns of bacteriophytochrome photoactivation.

Phytochromes are ubiquitous photosensor proteins, which control the growth, reproduction and movement in plants, fungi and bacteria. Phytochromes switch between two photophysical states depending on the light conditions. In analogy to molecular machines, light absorption induces a series of structural changes that are transduced from the bilin chromophore, through the protein, and to the output domains.

Signaling Mechanism of Phytochromes in Solution.

Phytochrome proteins guide the red/far-red photoresponse of plants, fungi, and bacteria. Crystal structures suggest that the mechanism of signal transduction from the chromophore to the output domains involves refolding of the so-called PHY tongue. It is currently not clear how the two other notable structural features of the phytochrome superfamily, the so-called helical spine and a knot in the peptide chain, are involved in photoconversion.

Quantum biology revisited.

Photosynthesis is a highly optimized process from which valuable lessons can be learned about the operating principles in nature. Its primary steps involve energy transport operating near theoretical quantum limits in efficiency. Recently, extensive research was motivated by the hypothesis that nature used quantum coherences to direct energy transfer.

The primary structural photoresponse of phytochrome proteins captured by a femtosecond X-ray laser.

Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation.

Transient IR spectroscopy identifies key interactions and unravels new intermediates in the photocycle of a bacterial phytochrome.

Phytochromes are photosensory proteins in plants, fungi, and bacteria, which detect red- and far-red light. They undergo a transition between the resting (Pr) and photoactivated (Pfr) states. In bacterial phytochromes, the Pr-to-Pfr transition is facilitated by two intermediate states, called Lumi-R and Meta-R.

Modulation of Structural Heterogeneity Controls Phytochrome Photoswitching.

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 radiodurans phytochrome changes from a structurally heterogeneous dark state to an ordered, light-activated state.

Ultrafast Chemical Exchange Dynamics of Hydrogen Bonds Observed via Isonitrile Infrared Sensors: Implications for Biomolecular Studies.

Local probes are indispensable to study protein structure and dynamics with site-specificity. The isonitrile functional group is a highly sensitive and H-bonding interaction-specific probe. Isonitriles exhibit large spectral shifts and transition dipole moment changes upon H-bonding while being weakly affected by solvent polarity.