Conformational dynamics are essential for proteins to function. We adapted time-resolved serial crystallography developed at x-ray lasers to visualize protein motions using synchrotrons. We recorded ...the structural changes in the light-driven proton-pump bacteriorhodopsin over 200 milliseconds in time. The snapshot from the first 5 milliseconds after photoactivation shows structural changes associated with proton release at a quality comparable to that of previous x-ray laser experiments. From 10 to 15 milliseconds onwards, we observe large additional structural rearrangements up to 9 angstroms on the cytoplasmic side. Rotation of leucine-93 and phenylalanine-219 opens a hydrophobic barrier, leading to the formation of a water chain connecting the intracellular aspartic acid-96 with the retinal Schiff base. The formation of this proton wire recharges the membrane pump with a proton for the next cycle.
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.
G-protein-coupled receptors (GPCRs) comprise the largest family of membrane proteins in the human genome and mediate cellular responses to an extensive array of hormones, neurotransmitters and ...sensory stimuli. Although some crystal structures have been determined for GPCRs, most are for modified forms, showing little basal activity, and are bound to inverse agonists or antagonists. Consequently, these structures correspond to receptors in their inactive states. The visual pigment rhodopsin is the only GPCR for which structures exist that are thought to be in the active state. However, these structures are for the apoprotein, or opsin, form that does not contain the agonist all-trans retinal. Here we present a crystal structure at a resolution of 3 Å for the constitutively active rhodopsin mutant Glu 113 Gln in complex with a peptide derived from the carboxy terminus of the α-subunit of the G protein transducin. The protein is in an active conformation that retains retinal in the binding pocket after photoactivation. Comparison with the structure of ground-state rhodopsin suggests how translocation of the retinal β-ionone ring leads to a rotation of transmembrane helix 6, which is the critical conformational change on activation. A key feature of this conformational change is a reorganization of water-mediated hydrogen-bond networks between the retinal-binding pocket and three of the most conserved GPCR sequence motifs. We thus show how an agonist ligand can activate its GPCR.
Serial crystallography was developed for the use at free-electron lasers but the approach has recently also been adapted to synchrotron sources. Here we discuss how the synergy between the two X-ray ...sources will facilitate a wide application of the technique in microcrystallography, room-temperature structure determination and time-resolved studies.
Acoustic levitation has attracted attention in terms of chemical and biochemical analysis in combination with various analytical methods because of its unique container-less environment for samples ...that is not reliant on specific material characteristics. However, loading samples with very high viscosity is difficult. To expand the scope, we propose the use of polymer thin films as sample holders, whereby the sample is dispensed on a film that is subsequently loaded onto an acoustic levitator. When applied for protein crystallography experiments, rotation controllability and positional stability are important prerequisites. We therefore study the acoustic levitation and rotation of thin films with an aspect ratio (the diameter-to-thickness ratio) of 80-240, which is an order of magnitude larger than those reported previously. For films with empirically optimized shapes, we find that it is possible to control the rotation speed in the range of 1-4 rotations per second while maintaining a positional stability of 12 ± 5 µm. The acoustic radiation force acting on the films is found to be a factor of 26-30 higher than that for same-volume water droplets. We propose use cases of the developed films for protein crystallography experiments and demonstrate data collections for large single crystal samples at room temperature.
Light-driven sodium pumps actively transport small cations across cellular membranes
. These pumps are used by microorganisms to convert light into membrane potential and have become useful ...optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved
, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser
, we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.
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.
Viral chemokine mimicry Standfuss, Jorg
Science (American Association for the Advancement of Science),
03/2015, Letnik:
347, Številka:
6226
Journal Article
Recenzirano
Chemokines are important regulators of the human immune system. Interactions of chemokines with their corresponding receptors guide white blood cells to the sites of infection, where they can trigger ...the required immune responses. Two complementary papers in this issue provide molecular details of these receptor-chemokine interactions. On page 1117, Qin et al. (1) report the crystal structure of the human CXCR4 receptor cross-linked to the viral chemokine vMIP-II. On page 1113, Burg et al. (2) describe the opposite case, the interaction of the human chemokine CX3CL1 with the viral US28 receptor (see the figure). Together, the studies provide crucial insights into the molecular mimicry that viruses use to fool the human immune system.
Optically controlled chemical reagents, termed “photopharmaceuticals,” are powerful tools for precise spatiotemporal control of proteins particularly when genetic methods, such as knockouts or ...optogenetics are not viable options. However, current photopharmaceutical scaffolds, such as azobenzenes are intolerant of GFP/YFP imaging and are metabolically labile, posing severe limitations for biological use. We rationally designed a photoswitchable "SBT" scaffold to overcome these problems, then derivatized it to create exceptionally metabolically robust and fully GFP/YFP-orthogonal "SBTub" photopharmaceutical tubulin inhibitors. Lead compound SBTub3 allows temporally reversible, cell-precise, and even subcellularly precise photomodulation of microtubule dynamics, organization, and microtubule-dependent processes. By overcoming the previous limitations of microtubule photopharmaceuticals, SBTubs offer powerful applications in cell biology, and their robustness and druglikeness are favorable for intracellular biological control in in vivo applications. We furthermore expect that the robustness and imaging orthogonality of the SBT scaffold will inspire other derivatizations directed at extending the photocontrol of a range of other biological targets.
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•SBTub3 photocontrols microtubule dynamics, organization, and dependent processes•Microtubule photocontrol is cell and sub-cellularly precise and temporally reversible•SBT photocontrol is orthogonal to GFP/YFP imaging and SBTs are metabolically stable•The SBT scaffold is promising for photopharmaceuticals for other protein targets
Photocontrollable reagents have unique potential as high spatiotemporal precision modulators of biological systems. Here, Gao et al. demonstrate a GFP-orthogonal and metabolically stable photoswitch that allows optical control over microtubule dynamics and architecture with subcellular resolution. The photoswitch scaffold also offers new possibilities for photopharmaceutical design against other targets.
The plant light‐harvesting complex of photosystem II (LHC‐II) collects and transmits solar energy for photosynthesis in chloroplast membranes and has essential roles in regulation of photosynthesis ...and in photoprotection. The 2.5 Å structure of pea LHC‐II determined by X‐ray crystallography of stacked two‐dimensional crystals shows how membranes interact to form chloroplast grana, and reveals the mutual arrangement of 42 chlorophylls a and b, 12 carotenoids and six lipids in the LHC‐II trimer. Spectral assignment of individual chlorophylls indicates the flow of energy in the complex and the mechanism of photoprotection in two close chlorophyll a–lutein pairs. We propose a simple mechanism for the xanthophyll‐related, slow component of nonphotochemical quenching in LHC‐II, by which excess energy is transferred to a zeaxanthin replacing violaxanthin in its binding site, and dissipated as heat. Our structure shows the complex in a quenched state, which may be relevant for the rapid, pH‐induced component of nonphotochemical quenching.
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