Search

Low-pass spectral analysis (LPSA) is a recently developed dynamics retrieval algorithm showing excellent retrieval properties when applied to model data affected by extreme incompleteness and stochastic weighting. In this work, we apply LPSA to an experimental time-resolved serial femtosecond crystallography (TR-SFX) dataset from the membrane protein bacteriorhodopsin (bR) and analyze its parametric sensitivity. While most dynamical modes are contaminated by nonphysical high-frequency features, we identify two dominant modes, which are little affected by spurious frequencies. The dynamics retrieved using these modes shows an isomerization signal compatible with previous findings. We employ synthetic data with increasing timing uncertainty, increasing incompleteness level, pixel-dependent incompleteness, and photon counting errors to investigate the root cause of the high-frequency contamination of our TR-SFX modes. By testing a range of methods, we show that timing errors comparable to the dynamical periods to be retrieved produce a smearing of dynamical features, hampering dynamics retrieval, but with no introduction of spurious components in the solution, when convergence criteria are met. Using model data, we are able to attribute the high-frequency contamination of low-order dynamical modes to the high levels of noise present in the data. Finally, we propose a method to handle missing observations that produces a substantial dynamics retrieval improvement from synthetic data with a significant static component. Reprocessing of the bR TR-SFX data using the improved method yields dynamical movies with strong isomerization signals compatible with previous findings. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Time-resolved serial crystallography captures snapshots of protein activation. • Dynamics in the fs to ms temporal regime can be visualized with near-atomic resolution. • Real-time information is critical to understand protein function. • High viscosity injectors reduce sample consumption. • Alternative trigger systems will widen the range of applications. Membrane proteins are the gatekeepers of cellular membranes where they act as enzymes, transporters, signaling receptors, or in energy conversion. Traditionally seen as a difficult field, the last decade has brought dramatic progress to membrane protein structural biology. Here, I describe recent advances in studying the conformational dynamics of membrane proteins by X-ray free electron lasers. By integrating sample efficient high viscosity injectors into pump probe setups, it has become possible to determine whole series of structural snapshots by time-resolved serial femtosecond crystallography and assemble them to molecular movies of proteins in action. According to the current studies on bacteriorhodopsin, photosystem II and nitric oxide reductase, I outline the technical challenges as well as new possibilities to study membrane protein function. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In the degenerative eye disease retinitis pigmentosa (RP), protein misfolding leads to fatal consequences for cell metabolism and rod and cone cell survival. To stop disease progression, a therapeutic approach focuses on stabilizing inherited protein mutants of the G protein-coupled receptor (GPCR) rhodopsin using pharmacological chaperones (PC) that improve receptor folding and trafficking. In this study, we discovered stabilizing nonretinal small molecules by virtual and thermofluor screening and determined the crystal structure of pharmacologically stabilized opsin at 2.4 Å resolution using one of the stabilizing hits (S-RS1). Chemical modification of S-RS1 and further structural analysis revealed the core binding motif of this class of rhodopsin stabilizers bound at the orthosteric binding site. Furthermore, previously unobserved conformational changes are visible at the intradiscal side of the seven-transmembrane helix bundle. A hallmark of this conformation is an open channel connecting the ligand binding site with the membrane and the intradiscal lumen of rod outer segments. Sufficient in size, the passage permits the exchange of hydrophobic ligands such as retinal. The results broaden our understanding of rhodopsin's conformational flexibility and enable therapeutic drug intervention against rhodopsin-related retinitis pigmentosa. [ABSTRACT FROM AUTHOR]

We present the main specifications of the newly constructed Swiss Free Electron Laser, SwissFEL, and explore its potential impact on ultrafast science. In light of recent achievements at current X-ray free electron lasers, we discuss the potential territory for new scientific breakthroughs offered by SwissFEL in Chemistry, Biology, and Materials Science, as well as nonlinear X-ray science. [ABSTRACT FROM AUTHOR]

Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin. In such an approach, microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser (XFEL) after a precise photoactivation delay. Here, a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from known vapour-diffusion crystallization protocols: (i) screening the low-salt crystallization conditions preferred for serial crystallography by vapour diffusion, (ii) optimization of batch crystallization, (iii) testing the crystal size and quality using second-harmonic generation (SHG) imaging and X-ray powder diffraction and (iv) production of millilitres of rhodopsin crystal suspension in batches for serial crystallography tests; these crystals diffracted at an XFEL at the Linac Coherent Light Source using a liquid-jet setup. [ABSTRACT FROM AUTHOR]

Arrestins function as adapter proteins that mediate G proteincoupled receptor (GPCR) desensitization, internalization, and additional rounds of signaling. Here we have compared binding of the GPCR rhodopsin to 403 mutants of arrestin-1 covering its complete sequence. This comprehensive and unbiased mutagenesis approach provides a functional dimension to the crystal structures of inactive, preactivated p44 and phosphopeptide-bound arrestins and will guide our understanding of arrestin-GPCR complexes. The presented functional map quantitatively connects critical interactions in the polar core and along the C tail of arrestin. A series of amino acids (Phe375, Phe377, Phe380, and Arg382) anchor the C tail in a position that blocks binding of the receptor. Interaction of phosphates in the rhodopsin C terminus with Arg29 controls a C-tail exchange mechanism in which the C tail of arrestin is released and exposes several charged amino acids (Lys14, Lys15, Arg18, Lys20, Lys110, and Lys300) for binding of the phosphorylated receptor C terminus. In addition to this arrestin phosphosensor, our data reveal several patches of amino acids in the finger (Gln69 and Asp73-Met75) and the lariat loops (L249-S252 and Y254) that can act as direct binding interfaces. A stretch of amino acids at the edge of the C domain (Trp194-Ser199, Gly337-Gly340, Thr343, and Thr345) could act as membrane anchor, binding interface for a second rhodopsin, or rearrange closer to the central loops upon complex formation. We discuss these interfaces in the context of experimentally guided docking between the crystal structures of arrestin and light-activated rhodopsin. [ABSTRACT FROM AUTHOR]

GPCRs (G-protein-coupled receptors) are seven-transmembrane helix proteins that transduce exogenous and endogenous signals to modulate the activity of downstream effectors inside the cell. Despite the relevance of these proteins in human physiology and pharmaceutical research, we only recently started to understand the structural basis of their activation mechanism. In the period 2008–2011, nine active-like structures of GPCRs were solved. Among them, we have determined the structure of light-activated rhodopsin with all the features of the active metarhodopsin-II, which represents so far the most native-like model of an active GPCR. This structure, together with the structures of other inactive, intermediate and active states of rhodopsin constitutes a unique structural framework on which to understand the conserved aspects of the activation mechanism of GPCRs. This mechanism can be summarized as follows: retinal isomerization triggers a series of local structural changes in the binding site that are amplified into three intramolecular activation pathways through TM (transmembrane helix) 5/TM3, TM6 and TM7/TM2. Sequence analysis strongly suggests that these pathways are conserved in other GPCRs. Differential activation of these pathways by ligands could be translated into the stabilization of different active states of the receptor with specific signalling properties. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCR) are seven transmembrane helix proteins that couple binding of extracellular ligands to conformational changes and activation of intracellular G proteins, GPCR kinases, and arrestins. Constitutively active mutants are ubiquitously found among GPCRs and increase the inherent basal activity of the receptor, which often correlates with a pathological outcome. Here, we have used the M257Y6.40 constitutively active mutant of the photoreceptor rhodopsin in combination with the specific binding of a C-terminal fragment from the G protein alpha subunit (GαCT) to trap a light activated state for crystallization. The structure of the M257Y/GαCT complex contains the agonist all-trans-retinal covalently bound to the native binding pocket and resembles the G protein binding metarhodopsin-II conformation obtained by the natural activation mechanism; i.e., illumination of the prebound chromophore 11-cis-retinal. The structure further suggests a molecular basis for the constitutive activity of 6.40 substitutions and the strong effect of the introduced tyrosine based on specific interactions with Y2235.58 in helix 5, Y3067.53 of the NPxxY motif and R1353.50 of the E(D)RY motif, highly conserved residues of the G protein binding site. [ABSTRACT FROM AUTHOR]

Recent years have seen tremendous breakthroughs in structure determination of G-protein-coupled receptors (GPCRs). In 2011, two agonist-bound active-state structures of rhodopsin have been published. Together with structures of several rhodopsin activation intermediates and a wealth of biochemical and spectroscopic information, they provide a unique structural framework on which to understand GPCR activation. Here we use this framework to compare the recent crystal structures of the agonist-bound active states of the β2 adrenergic receptor (β2AR) and the A2A adenosine receptor (A2AAR). While activation of these three GPCRs results in rearrangements of TM5 and TM6, the extent of this conformational change varies considerably. Displacements of the cytoplasmic side of TM6 ranges between 3 and 8Å depending on whether selective stabilizers of the active conformation are used (i.e. a G-protein peptide in the case of rhodopsin or a conformationally selective nanobody in the case of the β2AR) or not (A2AAR). The agonist-induced conformational changes in the ligand-binding pocket are largely receptor specific due to the different chemical nature of the agonists. However, several similarities can be observed, including a relocation of conserved residues W6.48 and F6.44 towards L5.51 and P5.50, and of I/L3.40 away from P5.50. This transmission switch links agonist binding to the movement of TM5 and TM6 through the rearrangement of the TM3–TM5–TM6 interface, and possibly constitutes a common theme of GPCR activation. [ABSTRACT FROM AUTHOR]

Abstract: Disruption of an interhelical salt bridge between the retinal protonated Schiff base linked to H7 and Glu113 on H3 is one of the decisive steps during activation of rhodopsin. Using previously established stabilization strategies, we engineered a stabilized E113Q counterion mutant that converted rhodopsin to a UV-absorbing photoreceptor with deprotonated Schiff base and allowed reconstitution into native-like lipid membranes. Fourier-transform infrared difference spectroscopy reveals a deprotonated Schiff base in the photoproducts of the mutant up to the active state Meta II, the absence of the classical pH-dependent Meta I/Meta II conformational equilibrium in favor of Meta II, and an anticipation of active state features under conditions that stabilize inactive photoproduct states in wildtype rhodopsin. Glu181 on extracellular loop 2, is found to be unable to maintain a counterion function to the Schiff base on the activation pathway of rhodopsin in the absence of the primary counterion, Glu113. The Schiff base becomes protonated in the transition to Meta III. This protonation is, however, not associated with a deactivation of the receptor, in contrast to wildtype rhodopsin. Glu181 is suggested to be the counterion in the Meta III state of the mutant and appears to be capable of stabilizing a protonated Schiff base in Meta III, but not of constraining the receptor in an inactive conformation. [Copyright &y& Elsevier]

Abstract: We determined the structure of the rhodopsin mutant N2C/D282C expressed in mammalian cells; the first structure of a recombinantly produced G protein-coupled receptor (GPCR). The mutant was designed to form a disulfide bond between the N terminus and loop E3, which allows handling of opsin in detergent solution and increases thermal stability of rhodopsin by 10 deg.C. It allowed us to crystallize a fully deglycosylated rhodopsin (N2C/N15D/D282C). N15 mutations are normally misfolding and cause retinitis pigmentosa in humans. Microcrystallographic techniques and a 5 μm X-ray beam were used to collect data along a single needle measuring 5 μm×5 μm×90 μm. The disulfide introduces only minor changes but fixes the N-terminal cap over the β-sheet lid covering the ligand-binding site, a likely explanation for the increased stability. This work allows structural investigation of rhodopsin mutants and shows the problems encountered during structure determination of GPCRs and other mammalian membrane proteins. [Copyright &y& Elsevier]

An introduction to the journal is presented in which the editor discusses the light-driven sodium pump used as next-generation inhibitory optogenetic tool.

The article discusses receptor-chemokine interaction in the human immune system, focusing on how viruses are able to mimic molecular structure, tricking the immune system. Topics include the results of two different studies presented in the same issue on the subject; G protein-coupled receptors (GPCR); and techniques for solving the structure of GPCR molecules.

The article focuses on a research which report the crystal structure of light-sensitive G protein coupled receptors (GPCRs) rhodopsin bound to an engineered mini-G protein. It mentions rhodopsin seems to adopt predominantly one thermodynamically stable active conformation and analysis of the well-defined GPCR–G protein interface suggests that the precise position of the carboxyl-terminal. It also mentions receptor is a significant determinant in selective G protein activation.

Photoisomerization of ligands is a key process in the field of photopharmacology. Thus, the kinetics and efficiency of this initial photoreaction are of great importance but can be influenced by the molecular environment of the binding pocket and the resulting confinement of the reaction pathway. In this study, we investigated the photoisomerization of an azobenzene derivative of the anti-Parkinson's drug istradefylline. To identify the impact of the binding pocket, the ligand was examined in solution and bound to its target protein, the A 2a adenosine receptor (A 2a R), belonging to the family of G protein-coupled receptors (GPCRs). Although the overall efficiency of isomerization is reduced when the ligand is bound, the initial photoreaction experiences little influence from the binding pocket. However, protein-coupled motion promotes a longer-lived excited-state population and thus leads to a reduction in efficiency. The results provide the kinetic basis for a photoswitchable GPCR ligand and demonstrate the influence of the binding pocket on fundamental photochemistry.