A5059555895- Photoreceptor and optogenetics research
- Retinal Development and Disorders
- Receptor Mechanisms and Signaling
- Thyroid Disorders and Treatments
- Advanced Fluorescence Microscopy Techniques
- Advanced X-ray Imaging Techniques
- Enzyme Structure and Function
- Advanced Electron Microscopy Techniques and Applications
- Neurobiology and Insect Physiology Research
- Mitochondrial Function and Pathology
- Mass Spectrometry Techniques and Applications
- Spectroscopy and Quantum Chemical Studies
- Photosynthetic Processes and Mechanisms
- Laser-Matter Interactions and Applications
- Neuroscience and Neuropharmacology Research
- Lipid Membrane Structure and Behavior
- Laser-Plasma Interactions and Diagnostics
- Erythrocyte Function and Pathophysiology
- Neural dynamics and brain function
- Thyroid Cancer Diagnosis and Treatment
- ATP Synthase and ATPases Research
- Ion channel regulation and function
- Antioxidant Activity and Oxidative Stress
- RNA and protein synthesis mechanisms
- Glaucoma and retinal disorders
Paul Scherrer Institute
2015-2024
European X-Ray Free-Electron Laser
2018
SLAC National Accelerator Laboratory
2018
Menlo School
2018
Swiss Nanoscience Institute
2018
Deutsches Elektronen-Synchrotron DESY
2018
University of Basel
2018
ETH Zurich
2015-2018
GEO Partner (Switzerland)
2018
Molecular Research Institute
2017
Look fast Organisms from bacteria to humans sense and react light. Proteins that contain the light-sensitive molecule retinal couple absorption of light conformational changes produce a signal or move ions across membrane. Nogly et al. used an x-ray laser probe earliest structural chromophore within microcrystals ion pump bacteriorhodopsin (see Perspective by Moffat). The excited-state wiggles but is held in place so only one double bond capable isomerizing. A water adjacent proton-pumping...
Abstract Historically, room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. Here, we demonstrate that serial millisecond crystallography at a synchrotron beamline equipped with high-viscosity injector and high frame-rate detector allows typical crystallographic experiments be performed room-temperature. Using crystal scanning approach, determine the high-resolution of sensitive molybdenum storage protein, soaking drug colchicine into...
Lipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins. Moreover, viscous LCP also provides a highly effective delivery medium serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, adaptation this technology to perform millisecond (SMX) more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates need difficult handling individual...
Abstract Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs) 1 . A photon absorbed 11- cis retinal chromophore rhodopsin, which isomerizes within 200 femtoseconds to all- trans conformation 2 , thereby initiating cellular signal transduction processes that ultimately lead vision. However, intramolecular mechanism photoactivated induces activation events inside remains experimentally unclear. Here we use ultrafast time-resolved crystallography at...
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 unidirectionality of have remained elusive. We combined time-resolved serial crystallography with spectroscopy multiscale simulations elucidate mechanism a chloride-pumping rhodopsin structural dynamics throughout cycle. traced transient anion-binding sites, obtained evidence how used in mechanism, identified steric...
We demonstrate absolute quantitative mass density mapping in three dimensions of frozen-hydrated biological matter with an isotropic resolution 180 nm. As model for a system we use Chlamydomonas cells buffer solution confined microcapillary. ptychographic X-ray computed tomography to image the entire specimen, including 18 μm-diameter capillary, thereby providing directly measurement uncertainty about 6%. The resulting maps have sufficient contrast distinguish from surrounding ice and...
Abstract Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and investigate protein dynamics modest sample requirement. Time-resolved (TR-SFX) delay 1 ms yields difference Fourier maps compatible dark M state transition bR. Importantly, very...
Light-sensitive G protein-coupled receptors (GPCRs)-rhodopsins-absorb photons to isomerize their covalently bound retinal, triggering conformational changes that result in downstream signaling cascades. Monostable rhodopsins release retinal upon isomerization as opposed the bistable "reisomerize" absorption of a second photon. Understanding mechanistic differences between these light-sensitive GPCRs has been hindered by scarcity recombinant models latter. Here, we reveal high-resolution...
For many scientific questions gaining three-dimensional insight into a specimen can provide valuable information. We here present an instrument called "tOMography Nano crYo (OMNY)," dedicated to high resolution 3D scanning x-ray microscopy at cryogenic conditions via hard X-ray ptychography. Ptychography is lens-less imaging method requiring accurate sample positioning. In OMNY, this in achieved laser interferometry and closed-loop position control reaching sub-10 nm positioning accuracy....
Precursor proteins of the solute carrier family and channel forming Tim components are imported into mitochondria in two main steps. First, they translocated through TOM complex outer membrane, a process assisted by Tim9/Tim10 complex. They passed on to TIM22 complex, which facilitates their insertion inner membrane. In present study, we have analyzed function translocation substrates across membrane mitochondria. The purified core was reconstituted lipid vesicles entrapped. precursor...
An experimental murine model of Graves' disease was used to produce monoclonal antibodies (mAbs) with thyroid stimulating activity. Two these, IRI-SAb2 and IRI-SAb3, showed particularly high potency (in the low nanomolar range) efficacy. behaved as a full agonist human TSH receptor (TSHr), even when tested in physiological salt concentrations. Both IRI-SAb3 were displaced from TSHr by autoantibodies patients or harboring thyroid-blocking antibodies, but not control subjects Hashimoto...
Structural information of the different conformational states two prototypical light-sensitive membrane proteins, bacteriorhodopsin and rhodopsin, has been obtained in past by X-ray cryo-crystallography cryo-electron microscopy. However, these methods do not allow for structure determination most intermediate conformations. Recently, potential X-Ray Free Electron Lasers (X-FELs) tracking dynamics light-triggered processes pump-probe serial femtosecond crystallography demonstrated using...
The amino-terminal ectodomain of the human TSH receptor has been expressed at surface CHO cells as a glycosylphosphatidylinositol-anchored molecule containing 10-residue histidine tag close to its C terminus. soluble could be released from by treatment with glycosylphosphatidylinositol−phospholipase and purified apparent homogeneity cobalt−Sepharose chromatography. Two nanomoles material was obtained, which suitable for analysis mass spectrometry. This allowed identification four out six...
Thyroid peroxidase (TPO1) is a membrane-bound heme-containing glycoprotein that catalyzes the synthesis of thyroid hormones. We generated stable cell lines expressing TPO1 and alternatively spliced isoform TPO2. Pulse-chase studies showed TPO2 half-life was dramatically decreased as compared with TPO1. The sensitivity to endo-β-N-acetylglucosaminidase H indicated protein processed through endoplasmic reticulum bears high mannose-type structures. Cell surface biotinylation experiments two...
Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, time-resolved serial femtosecond crystallography method required owing nonreversible activation of rhodopsin. such an approach, microcrystals in suspension are delivered into X-ray pulses free-electron laser (XFEL) after precise photoactivation delay. Here, millilitre batch...
The development of X-ray free-electron lasers (XFELs) has opened the possibility to investigate ultrafast dynamics biomacromolecules using diffraction. Whereas an increasing number structures solved by means serial femtosecond crystallography at XFELs is available, effect radiation damage on protein crystals during exposures remained open question. We used a split-and-delay line based diffractive optics Linac Coherent Light Source XFEL time dependence lysozyme crystals. For these tests, were...