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.

Aequorea's secrets revealed: New fluorescent proteins with unique properties for bioimaging and biosensing.

Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from A. victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.

Riboflavin-binding proteins for singlet oxygen production.

miniSOG, developed as the first fully genetically encoded singlet oxygen photosensitiser, has found various applications in cell imaging and functional studies. Yet, miniSOG has suboptimal properties, including a low yield of singlet oxygen generation, which can nevertheless be improved tenfold upon blue light irradiation. In a previous study, we showed that this improvement was due to the photolysis of the miniSOG chromophore, flavin mononucleotide (FMN), into lumichrome, with concomitant removal of the phosphoribityl tail, thereby improving oxygen access to the alloxazine ring. We thus reasoned that a chromophore with a shorter tail would readily improve the photosensitizing properties of miniSOG. In this work, we show that the replacement of FMN by riboflavin (RF), which lacks the bulky phosphate group, significantly improves the singlet oxygen quantum yield (ΦΔ). We then proceeded to mutagenize the residues stabilizing the phosphate group of FMN to alter the chromophore specificity. We identified miniSOG-R57Q as a flavoprotein that selectively binds RF in cellulo, with a modestly improved ΦΔ. Our results show that it is possible to modify the flavin specificity of a given flavoprotein, thus providing a new option to tune its photophysical properties, including those leading to photosensitization. We also determined the structure of miniSOG-Q103L, a mutant with a much increased ΦΔ, which allowed us to postulate the existence of another access channel to FMN for molecular oxygen.

Kinetic and Structural Characterization of the Self-Labeling Protein Tags HaloTag7, SNAP-tag, and CLIP-tag.

The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag, and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study of the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag, and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rate constants with certain rhodamine substrates, which are more than 2 orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rate constants, however, are less affected by the structure of the label than those of HaloTag7, which vary over 6 orders of magnitude for commonly employed substrates. Determining the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.

The Drosophila odorant-binding protein 28a is involved in the detection of the floral odour ß-ionone.

Odorant-binding proteins (OBPs) are small soluble proteins that are thought to transport hydrophobic odorants across the aqueous sensillar lymph to olfactory receptors. A recent study revealed that OBP28a, one of the most abundant Drosophila OBPs, is not required for odorant transport, but acts in buffering rapid odour variation in the odorant environment. To further unravel and decipher its functional role, we expressed recombinant OBP28a and characterized its binding specificity. Using a fluorescent binding assay, we found that OBP28a binds a restricted number of floral-like chemicals, including ß-ionone, with an affinity in the micromolar range. We solved the X-ray crystal structure of OBP28a, which showed extensive conformation changes upon ligand binding. Mutant flies genetically deleted for the OBP28a gene showed altered responses to ß-ionone at a given concentration range, supporting its essential role in the detection of specific compounds present in the natural environment of the fly.

Tracking the route of molecular oxygen in O2-tolerant membrane-bound [NiFe] hydrogenase.

[NiFe] hydrogenases catalyze the reversible splitting of H2 into protons and electrons at a deeply buried active site. The catalytic center can be accessed by gas molecules through a hydrophobic tunnel network. While most [NiFe] hydrogenases are inactivated by O2, a small subgroup, including the membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha, is able to overcome aerobic inactivation by catalytic reduction of O2 to water. This O2 tolerance relies on a special [4Fe3S] cluster that is capable of releasing two electrons upon O2 attack. Here, the O2 accessibility of the MBH gas tunnel network has been probed experimentally using a "soak-and-freeze" derivatization method, accompanied by protein X-ray crystallography and computational studies. This combined approach revealed several sites of O2 molecules within a hydrophobic tunnel network leading, via two tunnel entrances, to the catalytic center of MBH. The corresponding site occupancies were related to the O2 concentrations used for MBH crystal derivatization. The examination of the O2-derivatized data furthermore uncovered two unexpected structural alterations at the [4Fe3S] cluster, which might be related to the O2 tolerance of the enzyme.

Mechanistic Investigations of Green mEos4b Reveal a Dynamic Long-Lived Dark State.

Green-to-red photoconvertible fluorescent proteins (PCFPs) are key players in advanced microscopy schemes such as photoactivated localization microscopy (PALM). Whereas photoconversion and red-state blinking in PCFPs have been studied intensively, their green-state photophysical behavior has received less attention. Yet dark states in green PCFPs can become strongly populated in PALM schemes and exert an indirect but considerable influence on the quality of data recorded in the red channel. Furthermore, green-state photoswitching in PCFPs can be used directly for PALM and has been engineered to design highly efficient reversibly switchable fluorescent proteins (RSFPs) amenable to various nanoscopy schemes. Here, we demonstrate that green mEos4b efficiently switches to a long-lived dark state through cis-trans isomerization of its chromophore, as do most RSFPs. However, by combining kinetic crystallography, molecular dynamics simulations, and Raman spectroscopy, we find that the dark state in green mEos4b is much more dynamic than that seen in switched-off green IrisFP, a biphotochromic PCFP engineered from the common EosFP parent. Our data suggest that H-bonding patterns maintained by the chromophore in green PCFPs and RSFPs in both their on- and off-states collectively control photoswitching quantum yields. The reduced number of H-bonds maintained by the dynamic dark chromophore in green mEos4b thus largely accounts for the observed lower switching contrast as compared to that of IrisFP. We also compare the long-lived dark states reached from green and red mEos4b, on the basis of their X-ray structures and Raman signatures. Altogether, these data provide a unifying picture of the complex photophysics of PCFPs and RSFPs.

mScarlet: a bright monomeric red fluorescent protein for cellular imaging.

We report the engineering of mScarlet, a truly monomeric red fluorescent protein with record brightness, quantum yield (70%) and fluorescence lifetime (3.9 ns). We developed mScarlet starting with a consensus synthetic template and using improved spectroscopic screening techniques; mScarlet's crystal structure reveals a planar and rigidified chromophore. mScarlet outperforms existing red fluorescent proteins as a fusion tag, and it is especially useful as a Förster resonance energy transfer (FRET) acceptor in ratiometric imaging.

Chromophore Isomer Stabilization Is Critical to the Efficient Fluorescence of Cyan Fluorescent Proteins.

ECFP, the first usable cyan fluorescent protein (CFP), was obtained by adapting the tyrosine-based chromophore environment in green fluorescent protein to that of a tryptophan-based one. This first-generation CFP was superseded by the popular Cerulean, CyPet, and SCFP3A that were engineered by rational and random mutagenesis, yet the latter CFPs still exhibit suboptimal properties of pH sensitivity and reversible photobleaching behavior. These flaws were serendipitously corrected in the third-generation CFP mTurquoise and its successors without an obvious rationale. We show here that the evolution process had unexpectedly remodeled the chromophore environment in second-generation CFPs so they would accommodate a different isomer, whose formation is favored by acidic pH or light irradiation and which emits fluorescence much less efficiently. Our results illustrate how fluorescent protein engineering based solely on fluorescence efficiency optimization may affect other photophysical or physicochemical parameters and provide novel insights into the rational evolution of fluorescent proteins with a tryptophan-based chromophore.

Light-Oxygen-Voltage (LOV)-sensing Domains: Activation Mechanism and Optogenetic Stimulation.

The light-oxygen-voltage (LOV) domains of phototropins emerged as essential constituents of light-sensitive proteins, helping initiate blue light-triggered responses. Moreover, these domains have been identified across all kingdoms of life. LOV domains utilize flavin nucleotides as co-factors and undergo structural rearrangements upon exposure to blue light, which activates an effector domain that executes the final output of the photoreaction. LOV domains are versatile photoreceptors that play critical roles in cellular signaling and environmental adaptation; additionally, they can noninvasively sense and control intracellular processes with high spatiotemporal precision, making them ideal candidates for use in optogenetics, where a light signal is linked to a cellular process through a photoreceptor. The ongoing development of LOV-based optogenetic tools, driven by advances in structural biology, spectroscopy, computational methods, and synthetic biology, has the potential to revolutionize the study of biological systems and enable the development of novel therapeutic strategies.

Capturing the blue-light activated state of the Phot-LOV1 domain from Chlamydomonas reinhardtii using time-resolved serial synchrotron crystallography.

Light-oxygen-voltage (LOV) domains are small photosensory flavoprotein modules that allow the conversion of external stimuli (sunlight) into intracellular signals responsible for various cell behaviors (e.g. phototropism and chloroplast relocation). This ability relies on the light-induced formation of a covalent thioether adduct between a flavin chromophore and a reactive cysteine from the protein environment, which triggers a cascade of structural changes that result in the activation of a serine/threonine (Ser/Thr) kinase. Recent developments in time-resolved crystallography may allow the activation cascade of the LOV domain to be observed in real time, which has been elusive. In this study, we report a robust protocol for the production and stable delivery of microcrystals of the LOV domain of phototropin Phot-1 from Chlamydomonas reinhardtii (CrPhotLOV1) with a high-viscosity injector for time-resolved serial synchrotron crystallography (TR-SSX). The detailed process covers all aspects, from sample optimization to data collection, which may serve as a guide for soluble protein preparation for TR-SSX. In addition, we show that the crystals obtained preserve the photoreactivity using infrared spectroscopy. Furthermore, the results of the TR-SSX experiment provide high-resolution insights into structural alterations of CrPhotLOV1 from Δt = 2.5 ms up to Δt = 95 ms post-photoactivation, including resolving the geometry of the thioether adduct and the C-terminal region implicated in the signal transduction process.

Fixed-target pump-probe SFX: eliminating the scourge of light contamination.

X-ray free-electron laser (XFEL) light sources have enabled the rapid growth of time-resolved structural experiments, which provide crucial information on the function of macromolecules and their mechanisms. Here, the aim was to commission the SwissMX fixed-target sample-delivery system at the SwissFEL Cristallina experimental station using the PSI-developed micro-structured polymer (MISP) chip for pump-probe time-resolved experiments. To characterize the system, crystals of the light-sensitive protein light-oxygen-voltage domain 1 (LOV1) from Chlamydomonas reinhardtii were used. Using different experimental settings, the accidental illumination, referred to as light contamination, of crystals mounted in wells adjacent to those illuminated by the pump laser was examined. It was crucial to control the light scattering from and through the solid supports otherwise significant contamination occurred. However, the results here show that the opaque MISP chips are suitable for defined pump-probe studies of a light-sensitive protein. The experiment also probed the sub-millisecond structural dynamics of LOV1 and indicated that at Δt = 10 µs a covalent thioether bond is established between reactive Cys57 and its flavin mononucleotide cofactor. This experiment validates the crystals to be suitable for in-depth follow-up studies of this still poorly understood signal-transduction mechanism. Importantly, the fixed-target delivery system also permitted a tenfold reduction in protein sample consumption compared with the more common high-viscosity extrusion-based delivery system. This development creates the prospect of an increase in XFEL project throughput for the field.

Crystallization and preliminary X-ray diffraction analysis of the organophosphorus hydrolase OPHC2 from Pseudomonas pseudoalcaligenes.

Enzymes that are capable of degrading neurotoxic organophosphorus compounds are of increasing interest because of the lack of efficient and clean methods for their removal. Recently, a novel organophosphorus hydrolase belonging to the metallo-ß-lactamase superfamily was identified and isolated from the mesophilic bacterium Pseudomonas pseudoalcaligenes. This enzyme, named OPHC2, is endowed with significant thermal and pH stability, making it an appealing candidate for engineering studies to develop an efficient organophosphorus biodecontaminant. Combined with biochemical studies, structural information will help decipher the catalytic mechanism of organophosphorus hydrolysis by OPHC2 and identify the residues involved in its substrate specificity. Here, the expression, purification, crystallization and X-ray data collection at 2.1 Šresolution of OPHC2 are presented.

Crystallization and preliminary X-ray diffraction analysis of the lactonase VmoLac from Vulcanisaeta moutnovskia.

Phosphotriesterase-like lactonases (PLLs) are native lactonases that are capable of hydrolyzing lactones such as aliphatic lactones or acyl-homoserine lactones, which are involved in bacterial quorum sensing. Previously characterized PLLs are moreover endowed with a promiscuous phosphotriesterase activity and are therefore able to detoxify organophosphate insecticides. A novel PLL representative, dubbed VmoLac, has been identified from the hyperthermophilic crenarchaeon Vulcanisaeta moutnovskia. Because of its intrinsic high thermal stability, VmoLac may constitute an appealing candidate for engineering studies with the aim of producing an efficient biodecontaminant for organophosphorus compounds and a bacterial antivirulence agent. In combination with biochemical studies, structural information will allow the identification of the residues involved in substrate specificity and an understanding of the enzymatic catalytic mechanisms. Here, the expression, purification, crystallization and X-ray data collection at 2.4 Šresolution of VmoLac are reported.

Crystallization and preliminary X-ray diffraction analysis of a high-affinity phosphate-binding protein endowed with phosphatase activity from Pseudomonas aeruginosa PAO1.

In prokaryotes, phosphate starvation induces the expression of numerous phosphate-responsive genes, such as the pst operon including the high-affinity phosphate-binding protein (PBP or pstS) and alkaline phosphatases such as PhoA. This response increases the cellular inorganic phosphate import efficiency. Notably, some Pseudomonas species secrete, via a type-2 secretion system, a phosphate-binding protein dubbed LapA endowed with phosphatase activity. Here, the expression, purification, crystallization and X-ray data collection at 0.87 Šresolution of LapA are described. Combined with biochemical and enzymatic characterization, the structure of this intriguing phosphate-binding protein will help to elucidate the molecular origin of its phosphatase activity and to decipher its putative role in phosphate uptake.

Crystallization and preliminary X-ray diffraction analysis of a DING protein from Pseudomonas aeruginosa PA14.

DING proteins form an emergent family of proteins consisting of an increasing number of homologues that have been identified in all kingdoms of life. They belong to the superfamily of phosphate-binding proteins and exhibit a high affinity for phosphate. In eukaryotes, DING proteins have been isolated by virtue of their implication in several diseases and biological processes. Some of them are potent inhibitors of HIV-1 replication/transcription, raising the question of their potential involvement in the human defence system. Recently, a protein from Pseudomonas aeruginosa strain PA14, named PA14DING or LapC, belonging to the DING family has been identified. The structure of PA14DING, combined with detailed biochemical characterization and comparative analysis with available DING protein structures, will be helpful in understanding the structural determinants implicated in the inhibition of HIV-1 by DING proteins. Here, the expression, purification and crystallization of PA14DING and the collection of X-ray data to 1.9 Šresolution are reported.

Slow protein dynamics probed by time-resolved oscillation crystallography at room temperature.

The development of serial crystallography over the last decade at XFELs and synchrotrons has produced a renaissance in room-temperature macromolecular crystallography (RT-MX), and fostered many technical and methodological breakthroughs designed to study phenomena occurring in proteins on the picosecond-to-second timescale. However, there are components of protein dynamics that occur in much slower regimes, of which the study could readily benefit from state-of-the-art RT-MX. Here, the room-temperature structural study of the relaxation of a reaction intermediate at a synchrotron, exploiting a handful of single crystals, is described. The intermediate in question is formed in microseconds during the photoreaction of the LOV2 domain of phototropin 2 from Arabidopsis thaliana, which then decays in minutes. This work monitored its relaxation in the dark using a fast-readout EIGER X 4M detector to record several complete oscillation X-ray diffraction datasets, each of 1.2 s total exposure time, at different time points in the relaxation process. Coupled with in crystallo UV-Vis absorption spectroscopy, this RT-MX approach allowed the authors to follow the relaxation of the photoadduct, a thio-ether covalent bond between the chromophore and a cysteine residue. Unexpectedly, the return of the chromophore to its spectroscopic ground state is followed by medium-scale protein rearrangements that trigger a crystal phase transition and hinder the full recovery of the structural ground state of the protein. In addition to suggesting a hitherto unexpected role of a conserved tryptophan residue in the regulation of the photocycle of LOV2, this work provides a basis for performing routine time-resolved protein crystallography experiments at synchrotrons for phenomena occurring on the second-to-hour timescale.

Dynamics and mechanism of a light-driven chloride pump.

Chloride transport by microbial rhodopsins is an essential process for which molecular details such as the mechanisms that convert light energy to drive ion pumping and ensure the unidirectionality of the transport have remained elusive. We combined time-resolved serial crystallography with time-resolved spectroscopy and multiscale simulations to elucidate the molecular mechanism of a chloride-pumping rhodopsin and the structural dynamics throughout the transport cycle. We traced transient anion-binding sites, obtained evidence for how light energy is used in the pumping mechanism, and identified steric and electrostatic molecular gates ensuring unidirectional transport. An interaction with the π-electron system of the retinal supports transient chloride ion binding across a major bottleneck in the transport pathway. These results allow us to propose key mechanistic features enabling finely controlled chloride transport across the cell membrane in this light-powered chloride ion pump.

Structural insights and ab initio sequencing within the DING proteins family.

DING proteins constitute an intriguing family of phosphate-binding proteins that was identified in a wide range of organisms, from prokaryotes and archae to eukaryotes. Despite their seemingly ubiquitous occurrence in eukaryotes, their encoding genes are missing from sequenced genomes. Such a lack has considerably hampered functional studies. In humans, these proteins have been related to several diseases, like atherosclerosis, kidney stones, inflammation processes and HIV inhibition. The human phosphate binding protein is a human representative of the DING family that was serendipitously discovered from human plasma. An original approach was developed to determine ab initio the complete and exact sequence of this 38 kDa protein by utilizing mass spectrometry and X-ray data in tandem. Taking advantage of this first complete eukaryotic DING sequence, a immunohistochemistry study was undertaken to check the presence of DING proteins in various mice tissues, revealing that these proteins are widely expressed. Finally, the structure of a bacterial representative from Pseudomonas fluorescens was solved at sub-angstrom resolution, allowing the molecular mechanism of the phosphate binding in these high-affinity proteins to be elucidated.

Human-Phosphate-Binding-Protein inhibits HIV-1 gene transcription and replication.

The Human Phosphate-Binding protein (HPBP) is a serendipitously discovered lipoprotein that binds phosphate with high affinity. HPBP belongs to the DING protein family, involved in various biological processes like cell cycle regulation. We report that HPBP inhibits HIV-1 gene transcription and replication in T cell line, primary peripherical blood lymphocytes and primary macrophages. We show that HPBP is efficient in naïve and HIV-1 AZT-resistant strains. Our results revealed HPBP as a new and potent anti HIV molecule that inhibits transcription of the virus, which has not yet been targeted by HAART and therefore opens new strategies in the treatment of HIV infection.