Bacteriorhodopsin: Structural Insights Revealed Using X-Ray Lasers and Synchrotron Radiation.

Directional transport of protons across an energy transducing membrane-proton pumping-is ubiquitous in biology. Bacteriorhodopsin (bR) is a light-driven proton pump that is activated by a buried all-trans retinal chromophore being photoisomerized to a 13-cis conformation. The mechanism by which photoisomerization initiates directional proton transport against a proton concentration gradient has been studied by a myriad of biochemical, biophysical, and structural techniques. X-ray free electron lasers (XFELs) have created new opportunities to probe the structural dynamics of bR at room temperature on timescales from femtoseconds to milliseconds using time-resolved serial femtosecond crystallography (TR-SFX). Wereview these recent developments and highlight where XFEL studies reveal new details concerning the structural mechanism of retinal photoisomerization and proton pumping. We also discuss the extent to which these insights were anticipated by earlier intermediate trapping studies using synchrotron radiation. TR-SFX will open up the field for dynamical studies of other proteins that are not naturally light-sensitive.

Ultrafast structural changes direct the first molecular events of vision.

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.

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 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.

Spatiotemporal Release of Singlet Oxygen in Low Molecular Weight Organo-Gels Upon Thermal or Photochemical External Stimuli.

The first example of a material capable of spatiotemporal catch and release of singlet oxygen (1O2) in gel phase is presented. Several low molecular weight organogelators based around an oxotriphenylhexanoate (OTHO) core are developed and optimized with regard to; their gelation properties, and ability of releasing 1O2 upon thermal and/or photochemical external stimuli, in both gel phase and solution. Remarkably, reversible phase transitioning between the gel and solution phase are also demonstrated. Taken together two complementary modes of releasing 1O2, one thermally controlled over time, and one rapid release by means of photochemical stimuli is disclosed. These findings represent the first phase reversible system where function and aggregation properties can be controlled independently, and thus pave the way for novel applications in material sciences as well as in life sciences.

Characterization and evaluation of photolabile (µ-peroxo)(µ-hydroxo)bis[bis(bipyridyl)cobalt caged oxygen compounds to facilitate time-resolved crystallographic studies of cytochrome c oxidase.

Photolabile (µ-peroxo)(µ-hydroxo)bis[bis(bipyridyl)-cobalt-based caged oxygen compounds have been synthesized and characterized by optical absorbance spectroscopy, X-ray crystallography. and the quantum yield and redox stability were investigated. Furthermore, conditions were established where redox incompatibilities encountered between caged oxygen compounds and oxygen-dependant cytochrome c oxidase (CcO) could be circumvented. Herein, we demonstrate that millimolar concentrations of molecular oxygen can be released from a caged oxygen compound with spatio-temporal control upon laser excitation, triggering enzymatic turnover in cytochrome c oxidase. Spectroscopic evidence confirms the attainment of a homogeneous reaction initiation at concentrations and conditions relevant for further crystallography studies. This was demonstrated by the oxidizing microcrystals of reduced CcO by liberation of millimolar concentrations of molecular oxygen from a caged oxygen compound. We believe this will expand the scope of available techniques for the detailed investigation of oxygen-dependant enzymes with its native substrate and facilitate further time-resolved X-ray based studies such as wide/small angle X-ray scattering and serial femtosecond crystallography.

Nuclear envelope budding is a response to cellular stress.

Nuclear envelope budding (NEB) is a recently discovered alternative pathway for nucleocytoplasmic communication distinct from the movement of material through the nuclear pore complex. Through quantitative electron microscopy and tomography, we demonstrate how NEB is evolutionarily conserved from early protists to human cells. In the yeast Saccharomyces cerevisiae, NEB events occur with higher frequency during heat shock, upon exposure to arsenite or hydrogen peroxide, and when the proteasome is inhibited. Yeast cells treated with azetidine-2-carboxylic acid, a proline analog that induces protein misfolding, display the most dramatic increase in NEB, suggesting a causal link to protein quality control. This link was further supported by both localization of ubiquitin and Hsp104 to protein aggregates and NEB events, and the evolution of these structures during heat shock. We hypothesize that NEB is part of normal cellular physiology in a vast range of species and that in S. cerevisiae NEB comprises a stress response aiding the transport of protein aggregates across the nuclear envelope.

Transient isomers in the photodissociation of bromoiodomethane.

The photochemistry of halomethanes is fascinating for the complex cascade reactions toward either the parent or newly synthesized molecules. Here, we address the structural rearrangement of photodissociated CH2IBr in methanol and cyclohexane, probed by time-resolved X-ray scattering in liquid solution. Upon selective laser cleavage of the C-I bond, we follow the reaction cascade of the two geminate geometrical isomers, CH2I-Br and CH2Br-I. Both meta-stable isomers decay on different time scales, mediated by solvent interaction, toward the original parent molecule. We observe the internal rearrangement of CH2Br-I to CH2I-Br in cyclohexane by extending the time window up to 3 µs. We track the photoproduct kinetics of CH2Br-I in methanol solution where only one isomer is observed. The effect of the polarity of solvent on the geminate recombination pathways is discussed.

Visualizing a protein quake with time-resolved X-ray scattering at a free-electron laser.

We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis photosynthetic reaction center, observing an ultrafast global conformational change that arises within picoseconds and precedes the propagation of heat through the protein. This provides direct structural evidence for a 'protein quake': the hypothesis that proteins rapidly dissipate energy through quake-like structural motions.

Nanosecond pump-probe device for time-resolved serial femtosecond crystallography developed at SACLA.

X-ray free-electron lasers (XFELs) have opened new opportunities for time-resolved X-ray crystallography. Here a nanosecond optical-pump XFEL-probe device developed for time-resolved serial femtosecond crystallography (TR-SFX) studies of photo-induced reactions in proteins at the SPring-8 Angstrom Compact free-electron LAser (SACLA) is reported. The optical-fiber-based system is a good choice for a quick setup in a limited beam time and allows pump illumination from two directions to achieve high excitation efficiency of protein microcrystals. Two types of injectors are used: one for extruding highly viscous samples such as lipidic cubic phase (LCP) and the other for pulsed liquid droplets. Under standard sample flow conditions from the viscous-sample injector, delay times from nanoseconds to tens of milliseconds are accessible, typical time scales required to study large protein conformational changes. A first demonstration of a TR-SFX experiment on bacteriorhodopsin in bicelle using a setup with a droplet-type injector is also presented.

X-ray structure of human aquaporin 2 and its implications for nephrogenic diabetes insipidus and trafficking.

Human aquaporin 2 (AQP2) is a water channel found in the kidney collecting duct, where it plays a key role in concentrating urine. Water reabsorption is regulated by AQP2 trafficking between intracellular storage vesicles and the apical membrane. This process is tightly controlled by the pituitary hormone arginine vasopressin and defective trafficking results in nephrogenic diabetes insipidus (NDI). Here we present the X-ray structure of human AQP2 at 2.75 Å resolution. The C terminus of AQP2 displays multiple conformations with the C-terminal α-helix of one protomer interacting with the cytoplasmic surface of a symmetry-related AQP2 molecule, suggesting potential protein-protein interactions involved in cellular sorting of AQP2. Two Cd(2+)-ion binding sites are observed within the AQP2 tetramer, inducing a rearrangement of loop D, which facilitates this interaction. The locations of several NDI-causing mutations can be observed in the AQP2 structure, primarily situated within transmembrane domains and the majority of which cause misfolding and ER retention. These observations provide a framework for understanding why mutations in AQP2 cause NDI as well as structural insights into AQP2 interactions that may govern its trafficking.

Bacteriorhodopsin: Would the real structural intermediates please stand up?

BACKGROUND: Bacteriorhodopsin (bR) is the simplest known light driven proton pump and has been heavily studied using structural methods: eighty four X-ray diffraction, six electron diffraction and three NMR structures of bR are deposited within the protein data bank. Twenty one X-ray structures report light induced structural changes and changes induced by mutation, changes in pH, thermal annealing or X-ray induced photo-reduction have also been examined. SCOPE OF REVIEW: We argue that light-induced structural changes that are replicated across several studies by independent research groups are those most likely to represent what is happening in reality. We present both internal distance matrix analyses that sort deposited bR structures into hierarchal trees, and difference Fourier analysis of deposited X-ray diffraction data. MAJOR CONCLUSIONS: An internal distance matrix analysis separates most wild-type bR structures according to their different crystal forms, indicating how the protein's structure is influenced by crystallization conditions. A similar analysis clusters eleven studies of illuminated bR crystals as one branch of a hierarchal tree with reproducible movements of the extracellular portion of helix C towards helix G, and of the cytoplasmic portion of helix F away from helices A, B and G. All crystallographic data deposited for illuminated crystals show negative difference density on a water molecule (Wat402) that forms H-bonds to the retinal Schiff Base and two aspartate residues (Asp85, Asp212) in the bR resting state. Other recurring difference density features indicated reproducible side-chain, backbone and water molecule displacements. X-ray induced radiation damage also disorders Wat402 but acts via cleaving the head-groups of Asp85 and Asp212. GENERAL SIGNIFICANCE: A remarkable level of agreement exists when deposited structures and crystallographic observations are viewed as a whole. From this agreement a unified picture of the structural mechanism of light-induced proton pumping by bR emerges. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.

Appraising protein conformational changes by resampling time-resolved serial x-ray crystallography data.

With the development of serial crystallography at both x-ray free electron laser and synchrotron radiation sources, time-resolved x-ray crystallography is increasingly being applied to study conformational changes in macromolecules. A successful time-resolved serial crystallography study requires the growth of microcrystals, a mechanism for synchronized and homogeneous excitation of the reaction of interest within microcrystals, and tools for structural interpretation. Here, we utilize time-resolved serial femtosecond crystallography data collected from microcrystals of bacteriorhodopsin to compare results from partial occupancy structural refinement and refinement against extrapolated data. We illustrate the domain wherein the amplitude of refined conformational changes is inversely proportional to the activated state occupancy. We illustrate how resampling strategies allow coordinate uncertainty to be estimated and demonstrate that these two approaches to structural refinement agree within coordinate errors. We illustrate how singular value decomposition of a set of difference Fourier electron density maps calculated from resampled data can minimize phase bias in these maps, and we quantify residual densities for transient water molecules by analyzing difference Fourier and Polder omit maps from resampled data. We suggest that these tools may assist others in judging the confidence with which observed electron density differences may be interpreted as functionally important conformational changes.

Room-temperature serial synchrotron crystallography structure of Spinacia oleracea RuBisCO.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the enzyme responsible for the first step of carbon dioxide (CO2) fixation in plants, which proceeds via the carboxylation of ribulose 1,5-biphosphate. Because of the enormous importance of this reaction in agriculture and the environment, there is considerable interest in the mechanism of fixation of CO2 by RuBisCO. Here, a serial synchrotron crystallography structure of spinach RuBisCO is reported at 2.3 Šresolution. This structure is consistent with earlier single-crystal X-ray structures of this enzyme and the results are a good starting point for a further push towards time-resolved serial synchrotron crystallography in order to better understand the mechanism of the reaction.

No observable non-thermal effect of microwave radiation on the growth of microtubules.

Despite widespread public interest in the health impact of exposure to microwave radiation, studies of the influence of microwave radiation on biological samples are often inconclusive or contradictory. Here we examine the influence of microwave radiation of frequencies 3.5 GHz, 20 GHz and 29 GHz on the growth of microtubules, which are biological nanotubes that perform diverse functions in eukaryotic cells. Since microtubules are highly polar and can extend several micrometres in length, they are predicted to be sensitive to non-ionizing radiation. Moreover, it has been speculated that tubulin dimers within microtubules might rapidly toggle between different conformations, potentially participating in computational or other cooperative processes. Our data show that exposure to microwave radiation yields a microtubule growth curve that is distorted relative to control studies utilizing a homogeneous temperature jump. However, this apparent effect of non-ionizing radiation is reproduced by control experiments using an infrared laser or hot air to heat the sample and thereby mimic the thermal history of samples exposed to microwaves. As such, no non-thermal effects of microwave radiation on microtubule growth can be assigned. Our results highlight the need for appropriate control experiments in biophysical studies that may impact on the sphere of public interest.

Proteorhodopsin phototrophy promotes survival of marine bacteria during starvation.

Proteorhodopsins are globally abundant photoproteins found in bacteria in the photic zone of the ocean. Although their function as proton pumps with energy-yielding potential has been demonstrated, the ecological role of proteorhodopsins remains largely unexplored. Here, we report the presence and function of proteorhodopsin in a member of the widespread genus Vibrio, uncovered through whole-genome analysis. Phylogenetic analysis suggests that the Vibrio strain AND4 obtained proteorhodopsin through lateral gene transfer, which could have modified the ecology of this marine bacterium. We demonstrate an increased long-term survival of AND4 when starved in seawater exposed to light rather than held in darkness. Furthermore, mutational analysis provides the first direct evidence, to our knowledge, linking the proteorhodopsin gene and its biological function in marine bacteria. Thus, proteorhodopsin phototrophy confers a fitness advantage to marine bacteria, representing a novel mechanism for bacterioplankton to endure frequent periods of resource deprivation at the ocean's surface.

Light stimulates growth of proteorhodopsin-containing marine Flavobacteria.

Proteorhodopsins are bacterial light-dependent proton pumps. Their discovery within genomic material from uncultivated marine bacterioplankton caused considerable excitement because it indicated a potential phototrophic function within these organisms, which had previously been considered strictly chemotrophic. Subsequent studies established that sequences encoding proteorhodopsin are broadly distributed throughout the world's oceans. Nevertheless, the role of proteorhodopsins in native marine bacteria is still unknown. Here we show, from an analysis of the complete genomes of three marine Flavobacteria, that cultivated bacteria in the phylum Bacteroidetes, one of the principal components of marine bacterioplankton, contain proteorhodopsin. Moreover, growth experiments in both natural and artificial seawater (low in labile organic matter, which is typical of the world's oceans) establish that exposure to light results in a marked increase in the cell yield of one such bacterium (Dokdonia sp. strain MED134) when compared with cells grown in darkness. Thus, our results show that the phototrophy conferred by proteorhodopsin can provide critical amounts of energy, not only for respiration and maintenance but also for active growth of marine bacterioplankton in their natural environment.

A simple goniometer-compatible flow cell for serial synchrotron X-ray crystallography.

Serial femtosecond crystallography was initially developed for room-temperature X-ray diffraction studies of macromolecules at X-ray free electron lasers. When combined with tools that initiate biological reactions within microcrystals, time-resolved serial crystallography allows the study of structural changes that occur during an enzyme catalytic reaction. Serial synchrotron X-ray crystallography (SSX), which extends serial crystallography methods to synchrotron radiation sources, is expanding the scientific community using serial diffraction methods. This report presents a simple flow cell that can be used to deliver microcrystals across an X-ray beam during SSX studies. This device consists of an X-ray transparent glass capillary mounted on a goniometer-compatible 3D-printed support and is connected to a syringe pump via light-weight tubing. This flow cell is easily mounted and aligned, and it is disposable so can be rapidly replaced when blocked. This system was demonstrated by collecting SSX data at MAX IV Laboratory from microcrystals of the integral membrane protein cytochrome c oxidase from Thermus thermophilus, from which an X-ray structure was determined to 2.12 Šresolution. This simple SSX platform may help to lower entry barriers for non-expert users of SSX.

Time-resolved serial crystallography to track the dynamics of carbon monoxide in the active site of cytochrome c oxidase.

Cytochrome c oxidase (CcO) is part of the respiratory chain and contributes to the electrochemical membrane gradient in mitochondria as well as in many bacteria, as it uses the energy released in the reduction of oxygen to pump protons across an energy-transducing biological membrane. Here, we use time-resolved serial femtosecond crystallography to study the structural response of the active site upon flash photolysis of carbon monoxide (CO) from the reduced heme a3 of ba3-type CcO. In contrast with the aa3-type enzyme, our data show how CO is stabilized on CuB through interactions with a transiently ordered water molecule. These results offer a structural explanation for the extended lifetime of the CuB-CO complex in ba3-type CcO and, by extension, the extremely high oxygen affinity of the enzyme.

Glycosylation increases the thermostability of human aquaporin 10 protein.

Human aquaporin10 (hAQP10) is a transmembrane facilitator of both water and glycerol transport in the small intestine. This aquaglyceroporin is located in the apical membrane of enterocytes and is believed to contribute to the passage of water and glycerol through these intestinal absorptive cells. Here we overproduced hAQP10 in the yeast Pichia pastoris and observed that the protein is glycosylated at Asn-133 in the extracellular loop C. This finding confirms one of three predicted glycosylation sites for hAQP10, and its glycosylation is unique for the human aquaporins overproduced in this host. Nonglycosylated protein was isolated using both glycan affinity chromatography and through mutating asparagine 133 to a glutamine. All three forms of hAQP10 where found to facilitate the transport of water, glycerol, erythritol, and xylitol, and glycosylation had little effect on functionality. In contrast, glycosylated hAQP10 showed increased thermostability of 3-6 °C compared with the nonglycosylated protein, suggesting a stabilizing effect of the N-linked glycan. Because only one third of hAQP10 was glycosylated yet the thermostability titration was mono-modal, we suggest that the presence of at least one glycosylated protein within each tetramer is sufficient to convey an enhanced structural stability to the remaining hAQP10 protomers of the tetramer.