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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 JÉ 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.
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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.
Asunto(s)
Desoxirribodipirimidina Fotoliasa , Humanos , Animales , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Triptófano/química , Electrones , Drosophila melanogaster/metabolismo , Escherichia coli/genética , Transporte de Electrón , Cristalografía por Rayos XRESUMEN
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. For liquid samples, the solution typically involves some form of high-speed liquid jet, capable of keeping up with the rate of X-ray pulses. However, conventional jets are not ideal because of radiation-induced explosions of the jet, as well as their cylindrical geometry combined with the X-ray pointing instability of many beamlines which causes the interaction volume to differ for every pulse. This complicates data analysis and contributes to measurement errors. An alternative geometry is a liquid sheet jet which, with its constant thickness over large areas, eliminates the problems related to X-ray pointing. Since liquid sheets can be made very thin, the radiation-induced explosion is reduced, boosting their stability. These are especially attractive for experiments which benefit from small interaction volumes such as fluctuation X-ray scattering and several types of spectroscopy. Although their use has increased for soft X-ray applications in recent years, there has not yet been wide-scale adoption at XFELs. Here, gas-accelerated liquid sheet jet sample injection is demonstrated at the European XFEL SPB/SFX nano focus beamline. Its performance relative to a conventional liquid jet is evaluated and superior performance across several key factors has been found. This includes a thickness profile ranging from hundreds of nanometres to 60â nm, a fourfold increase in background stability and favorable radiation-induced explosion dynamics at high repetition rates up to 1.13â MHz. Its minute thickness also suggests that ultrafast single-particle solution scattering is a possibility.
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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.
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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. In this study, we apply isotope-labeling techniques to isolate the vibrational bands from the protein-bound chromophore of the bacterial phytochrome from Deinococcus radiodurans. We elucidate the structure and ultrafast dynamics of the chromophore with 2D infra-red (IR) spectroscopy and molecular dynamics simulations. The carbonyl stretch vibrations of the pyrrole rings show the heterogeneous distribution of hydrogen-bonding structures, which exhibit distinct ultrafast relaxation dynamics. Moreover, we resolve a previously undetected 1678 cm-1 band that is strongly coupled to the A- and D-ring of biliverdin and demonstrate the presence of complex vibrational redistribution pathways between the biliverdin modes with relaxation-assisted measurements of 2D IR cross peaks. In summary, we expect 2D IR spectroscopy to be useful in explaining how point mutations in the protein sequence affect the hydrogen-bonding structure around the chromophore and consequently its ability to photoisomerize to the light-activated states.
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Biliverdina , Fitocromo , Vibración , Espectrofotometría Infrarroja , HidrógenoRESUMEN
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. We reveal cryo electron microscopy structures of the complete phytochrome from Deinoccocus radiodurans in its resting and photoactivated states at 3.6 Å and 3.5 Å resolution, respectively. Upon photoactivation, the photosensory core module hardly changes its tertiary domain arrangement, but the connector helices between the photosensory and the histidine kinase modules open up like a zipper, causing asymmetry and disorder in the effector domains. The structures provide a framework for atom-scale understanding of signaling in phytochromes, visualize allosteric communication over several nanometers, and suggest that disorder in the dimeric arrangement of the effector domains is important for phosphatase activity in a two-component system. The results have implications for the development of optogenetic applications.
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Fitocromo , Fitocromo/metabolismo , Histidina Quinasa/metabolismo , Modelos Moleculares , Proteínas Bacterianas/metabolismo , Transducción de Señal , Luz , Bacterias/metabolismoRESUMEN
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. In this review, we summarize recent advances on detecting structural changes for phytochrome photosensor proteins and a bacterial photosynthetic reaction center. In the phytochrome structural changes are extensive and involve major rearrangements of many amino acids and water molecules, accompanying the regulation of its biochemical activity, whereas in the photosynthetic reaction center protein the structural changes are smaller, more localized, and are optimized to facilitate electron transfer along the chromophores. The detected structural motions underpin the proteins' function, providing a showcase for the importance of detecting ultrafast protein structural dynamics.
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Proteínas del Complejo del Centro de Reacción Fotosintética , Fitocromo , Cristalografía por Rayos X , Conformación Proteica , Proteínas/química , Cristalografía , Fitocromo/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismoRESUMEN
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 structure explains how the protein environment stabilizes the radical pair state, which is crucial for function of (6-4) photolyases and cryptochromes.
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Desoxirribodipirimidina Fotoliasa , Aminoácidos , Animales , Criptocromos/química , Criptocromos/metabolismo , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/metabolismo , Flavina-Adenina Dinucleótido/química , Triptófano/químicaRESUMEN
(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. The room-temperature structure was solved to 2.27â Å resolution and was obtained by serial femtosecond crystallography (SFX) using an X-ray free-electron laser. The crystallization and preparation conditions are also reported. The cryogenic structure was solved to 1.79â Å resolution using conventional X-ray crystallography. The structures agree with each other, indicating that the structural information obtained from crystallography at cryogenic temperature also applies at room temperature. Furthermore, UV-Vis absorption spectroscopy confirms that Dm(6-4)photolyase is photoactive in the crystals, giving a green light to time-resolved SFX studies on the protein, which can reveal the structural mechanism of the photoactivated protein in DNA repair.
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Flavoproteínas/química , Animales , Cristalografía , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/metabolismo , Drosophila melanogaster , Flavoproteínas/metabolismo , TemperaturaRESUMEN
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. Here, we resolve this long-standing conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome from Agrobacterium fabrum (Agp1). Whereas Agp1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While Agp1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes suggest the use of light-controllable histidine kinases and phosphatases for optogenetics.
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Proteínas Bacterianas/metabolismo , Histidina Quinasa/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fotorreceptores Microbianos/metabolismo , Transducción de Señal/efectos de la radiación , Agrobacterium/enzimología , Proteínas Bacterianas/ultraestructura , Deinococcus/enzimología , Histidina Quinasa/ultraestructura , Luz , Simulación de Dinámica Molecular , Monoéster Fosfórico Hidrolasas/ultraestructura , Fotorreceptores Microbianos/ultraestructura , Dominios ProteicosRESUMEN
Phytochrome photoreceptors regulate vital adaptations of plant development, growth, and physiology depending on the ratio of red and far-red light. The light-triggered Z/E 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. To address this deficit, we studied the Arabidopsis thaliana phytochrome A (AtphyA) at full length by cryo-electron microscopy (cryo-EM). Following heterologous expression in Escherichia coli, we optimized the solvent conditions to overcome protein aggregation and thus obtained photochemically active, near-homogenous AtphyA. We prepared grids for cryo-EM analysis of AtphyA in its Pr state and conducted single-particle analysis. The resulting two-dimensional class averages and the three-dimensional electron density map at 17 Å showed a homodimeric head-to-head assembly of AtphyA. Docking of domain structures into the electron density revealed a separation of the AtphyA homodimer at the junction of its photosensor and effector modules, as reflected in a large void in the middle of map. The overall architecture of AtphyA resembled that of bacterial phytochromes, thus hinting at commonalities in signal transduction and mechanism between these receptors. Our work paves the way toward future studies of the structure, light response, and interactions of full-length phytochromes by cryo-EM.
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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. It is unknown how the Pr-to-Pfr interconversion occurs, as the structure of intermediates remains notoriously elusive. Here, we present short-lived crystal structures of the photosensory core modules of the bacteriophytochrome from myxobacterium Stigmatella aurantiaca captured by an X-ray free electron laser 5 ns and 33 ms after light illumination of the Pr state. We observe large structural displacements of the covalently bound bilin chromophore, which trigger a bifurcated signaling pathway that extends through the entire protein. The snapshots show with atomic precision how the signal progresses from the chromophore, explaining how plants, bacteria, and fungi sense red light.
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Fitocromo/química , Fitocromo/metabolismo , Stigmatella aurantiaca/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Conformación ProteicaRESUMEN
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. Whereas the derivative shape of the band-shift signal is well understood in transient absorption spectroscopy, its emergence in two-dimensional electronic spectroscopy (2DES) has not been discussed. In this work, we employed 2DES to follow the dynamic band-shift signal in reaction centers of purple bacteria Rhodobacter sphaeroides at 77 K. The prominent two-dimensional derivative-shape signal appears with the characteristic formation time of the charge separated state. To explain and characterize the band-shift signal, we use expanded double-sided Feynman diagram formalism. We propose to distinguish two types of Feynman diagrams that lead to signals with negative amplitude: excited state absorption and re-excitation. The presented signal decomposition and modeling analysis allows us to recover precise electrochromic shifts of accessory bacteriochlorophylls, identify additional signals in the B band range, and gain a further insight into the electron transfer mechanism. In a broader perspective, expanded Feynman diagram formalism will allow for interpretation of all 2D signals in a clearer and more intuitive way and therefore facilitate studying the underlying photophysics.
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Electrones , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Rhodobacter sphaeroides/enzimología , Análisis Espectral , Proteínas del Complejo del Centro de Reacción Fotosintética/químicaRESUMEN
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. Here, we present solution NMR data of the complete photosensory core module from Deinococcus radiodurans. Photoswitching between the resting and the active states induces changes in amide chemical shifts, residual dipolar couplings, and relaxation dynamics. All observables indicate a photoinduced structural change in the knot region and lower part of the helical spine. This implies that a conformational signal is transduced from the chromophore to the helical spine through the PAS and GAF domains. The discovered pathway underpins functional studies of plant phytochromes and may explain photosensing by phytochromes under biological conditions.
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Proteínas Bacterianas/química , Fitocromo/química , Proteínas Bacterianas/metabolismo , Deinococcus/química , Simulación de Dinámica Molecular , Fitocromo/metabolismo , Conformación Proteica en Hélice alfa , Transducción de SeñalRESUMEN
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 crystallography1 using an X-ray free-electron laser2 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. 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.
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Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Bacterioclorofilas/metabolismo , Sitios de Unión/efectos de los fármacos , Clorofila/metabolismo , Clorofila/efectos de la radiación , Cristalografía , Citoplasma/metabolismo , Transporte de Electrón/efectos de los fármacos , Electrones , Hyphomicrobiaceae/enzimología , Hyphomicrobiaceae/metabolismo , Rayos Láser , Modelos Moleculares , Oxidación-Reducción/efectos de la radiación , Feofitinas/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/efectos de la radiación , Protones , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo , Vitamina K 2/metabolismoRESUMEN
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. We extract how translational and rotational kinetic energy is dispersed into the solvent. We also find that 85% of the primary radical pairs are confined to their original solvent cage and discuss how this influences the downstream recombination reactions.
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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. We suggest an approach using the coupling deconvolution in the framework of compressed sensing (CS) spectra processing that leads to a major increase in resolution, sensitivity, and overall quality of NUS reconstruction. A new mathematical description of the decoupling by deconvolution explains the effects of thermal noise and reveals a relation with the underlying assumption of the CS. The gain in resolution and sensitivity for challenging molecular systems is demonstrated for the key HNCA experiment used for protein backbone assignment applied to two large proteins: intrinsically disordered 441-residue Tau and a 509-residue globular bacteriophytochrome fragment. The approach will be valuable in a multitude of chemistry applications, where NMR experiments are compromised by the homonuclear scalar coupling.
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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. Recent progress towards understanding this structural mechanism of signal transduction has been manifold. We describe this progress with a focus on bacteriophytochromes. We describe the mechanism along three structural tiers, which are the chromophore-binding pocket, the photosensory module, and the output domains. We discuss possible interconnections between the tiers and conclude by presenting future directions and open questions. We hope that this review may serve as a compendium to guide future structural and spectroscopic studies designed to understand structural signaling in phytochromes.
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Proteínas Bacterianas/química , Fitocromo/química , Proteínas Bacterianas/metabolismo , Modelos Moleculares , Procesos Fotoquímicos , Fitocromo/metabolismo , Conformación Proteica , Transducción de SeñalRESUMEN
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. This body of work, a cornerstone for the field of quantum biology, rests on the interpretation of small-amplitude oscillations in two-dimensional electronic spectra of photosynthetic complexes. This Review discusses recent work reexamining these claims and demonstrates that interexciton coherences are too short lived to have any functional significance in photosynthetic energy transfer. Instead, the observed long-lived coherences originate from impulsively excited vibrations, generally observed in femtosecond spectroscopy. These efforts, collectively, lead to a more detailed understanding of the quantum aspects of dissipation. Nature, rather than trying to avoid dissipation, exploits it via engineering of exciton-bath interaction to create efficient energy flow.