Charakterisierung allosterischer Bindungsstellen von P2X4-Rezeptor-Modulatoren

P2X-Rezeptoren stehen aufgrund ihrer Beteiligung an zahlreichen (patho)physiologischen Prozessen wie der Wahrnehmung von Schmerz, entzündlichen Veränderungen oder Krebs als Zielstrukturen für potentielle neue Wirkstoffe im Fokus der Forschung. Der P2X4-Rezeptor gilt aufgrund seiner hohen Expression in spinalen Microglia-Zellen, die mit neuropathischem Schmerz assoziiert werden, als vielversprechende Arzneimittel-Zielstruktur. Die Blockade sowie der Knockout von P2X4-Rezeptoren führten zu deutlich reduzierten neuropathischen Schmerzzuständen. Die Aktivierung von P2X4-Rezeptoren gilt dagegen als neue Strategie in der Therapie der Alkoholabhängigkeit. Ethanol wirkt als allosterischer Antagonist des P2X4-Rezeptors und der Missbrauch von Alkohol führte im Ratten-Modell zu einer reduzierten P2X4-Rezeptor-Genexpression. Ziel der vorliegenden Arbeit war es, die Mechanismen der allosterischen Modulation des P2X4-Rezeptors zu verstehen und die relevante(n) Bindungsstelle(n) bekannter und neuer allosterischer Modulatoren zu identifizieren, um damit eine verbesserte Basis für die Entwicklung neuer, potenter und selektiver positiver und negativer allosterischer P2X4-Rezeptor-Modulatoren (PAMs und NAMs) zu schaffen. Durch Herstellung und Untersuchung von P2X4(P2X2)-Rezeptorchimären und mit Hilfe von zielgerichteter Mutagenese ist es gelungen, allosterische Bindungsstellen für bekannte und neue positive und negative allosterische Modulatoren (PAMs und NAMs) des humanen P2X4-Rezeptors zu identifizieren. Die vorliegenden Ergebnisse deuten auf die Existenz mehrerer allosterischer Bindungsstellen im Bereich der extrazellulären Domäne des humanen P2X4-Rezeptors hin, was die Vielseitigkeit der P2X-Rezeptoren als "Drug Targets" verdeutlicht. Die erhaltenen Ergebnisse können dazu beitragen, die Entwicklung neuer Arzneistoffe, nicht nur für P2X4-Rezeptoren, sondern darüber hinaus für weitere P2X-Rezeptor-Subtypen entscheidend voranzutreiben

Peptide model helices in lipid membranes: insertion, positioning, and lipid response on aggregation studied by X-ray scattering

Studying membrane active peptides or protein fragments within the lipid bilayer environment is particularly challenging in the case of synthetically modified, labeled, artificial, or recently discovered native structures. For such samples the localization and orientation of the molecular species or probe within the lipid bilayer environment is the focus of research prior to an evaluation of their dynamic or mechanistic behavior. X-ray scattering is a powerful method to study peptide/lipid interactions in the fluid, fully hydrated state of a lipid bilayer. For one, the lipid response can be revealed by observing membrane thickening and thinning as well as packing in the membrane plane; at the same time, the distinct positions of peptide moieties within lipid membranes can be elucidated at resolutions of up to several angstroms by applying heavy-atom labeling techniques. In this study, we describe a generally applicable X-ray scattering approach that provides robust and quantitative information about peptide insertion and localization as well as peptide/lipid interaction within highly oriented, hydrated multilamellar membrane stacks. To this end, we have studied an artificial, designed β-helical peptide motif in its homodimeric and hairpin variants adopting different states of oligomerization. These peptide lipid complexes were analyzed by grazing incidence diffraction (GID) to monitor changes in the lateral lipid packing and ordering. In addition, we have applied anomalous reflectivity using synchrotron radiation as well as in-house X-ray reflectivity in combination with iodine-labeling in order to determine the electron density distribution ρ(z) along the membrane normal (z axis), and thereby reveal the hydrophobic mismatch situation as well as the position of certain amino acid side chains within the lipid bilayer. In the case of multiple labeling, the latter technique is not only applicable to demonstrate the peptide’s reconstitution but also to generate evidence about the relative peptide orientation with respect to the lipid bilayer

Heterogeneous and self-organizing mineralization of bone matrix promoted by hydroxyapatite nanoparticles

The mineralization process is crucial to the load-bearing characteristics of the bone extracellular matrix. In this work, we have studied the spatiotemporal dynamics of mineral deposition by human bone marrow mesenchymal stem cells differentiating toward osteoblasts promoted by the presence of exogenous hydroxyapatite nanoparticles. At molecular level, the added nanoparticles positively modulated the expression of bone-specific markers and enhanced calcified matrix deposition during osteogenic differentiation. The nucleation, growth and spatial arrangement of newly deposited hydroxyapatite nanocrystals have been evaluated using Scanning Micro X-Ray Diffraction and Scanning Micro X-Ray Fluorescence. As leading results, we have found the emergence of a complex scenario where the spatial organization and temporal evolution of the process exhibit a heterogeneous and self-organizing dynamics. At the same time the possibility to control the differentiation kinetic through the addition of synthetic nanoparticles, paves the way to empower the generation of more structured bone scaffolds in tissue engineering and to design new drugs in regenerative medicine

In Situ Ptychography of Heterogeneous Catalysts using Hard X-Rays: High Resolution Imaging at Ambient Pressure and Elevated Temperature

A new closed cell is presented for in situ X-ray ptychography which allows studies under gas flow and at elevated temperature. In order to gain complementary information by transmission and scanning electron microscopy, the cell makes use of a Protochips E-chipTM which contains a small, thin electron transparent window and allows heating. Two gold-based systems, 50 nm gold particles and nanoporous gold as a relevant catalyst sample, were used for studying the feasibility of the cell. Measurements showing a resolution around 40 nm have been achieved under a flow of synthetic air and during heating up to temperatures of 933 K. An elevated temperature exhibited little influence on image quality and resolution. With this study, the potential of in situ hard X-ray ptychography for investigating annealing processes of real catalyst samples is demonstrated. Furthermore, the possibility to use the same sample holder for ex situ electron microscopy before and after the in situ study underlines the unique possibilities available with this combination of electron microscopy and X-ray microscopy on the same sample

Megahertz pulse trains enable multi-hit serial femtosecond crystallography experiments at X-ray free electron lasers

The European X-ray Free Electron Laser (XFEL) and Linac Coherent Light Source (LCLS) II are extremely intense sources of X-rays capable of generating Serial Femtosecond Crystallography (SFX) data at megahertz (MHz) repetition rates. Previous work has shown that it is possible to use consecutive X-ray pulses to collect diffraction patterns from individual crystals. Here, we exploit the MHz pulse structure of the European XFEL to obtain two complete datasets from the same lysozyme crystal, first hit and the second hit, before it exits the beam. The two datasets, separated by <1 µs, yield up to 2.1 Å resolution structures. Comparisons between the two structures reveal no indications of radiation damage or significant changes within the active site, consistent with the calculated dose estimates. This demonstrates MHz SFX can be used as a tool for tracking sub-microsecond structural changes in individual single crystals, a technique we refer to as multi-hit SFX

Segmented flow generator for serial crystallography at the European X-ray free electron laser

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported

Mapping Morphological and Structural Properties of Lead Halide Perovskites by Scanning Nanofocus XRD

Scanning nanofocus X-ray diffraction (nXRD) performed at a synchrotron is used to simultaneously probe the morphology and the structural properties of spin-coated CH$_3$NH$_3$PbI$_3$ (MAPI) perovskite films for photovoltaic devices. MAPI films are spin-coated on a Si/SiO$_2$/poly(3,4-ethylenedioxythiophene): polystyrene sulfonate (PEDOT:PSS) substrate held at different temperatures during the deposition in order to tune the perovskite film coverage. The films are then investigated using nXRD and scanning electron microscopy (SEM). The advantages of nXRD over SEM and other techniques are discussed. A method to visualize, selectively isolate, and structurally characterize single perovskite grains buried within a complex, polycrystalline film is developed. The results of nXRD measurements are correlated with solar cell device measurements, and it is shown that spin-coating the perovskite precursor solution at elevated temperatures leads to improved surface coverage and enhanced solar cell performance.This work was funded by the UK Engineering and Physical Sciences Research Council via grants EP/M025020/1 “High resolution mapping of performance and degradation mechanisms in printable photovoltaic devices,” EP/J017361/1 (Supersolar Solar Energy Hub) and the E-Futures Doctoral Training Center in Interdisciplinary Energy Research EP/G037477/1. This work was partially funded by the President of the UAE’s Distinguished Student Scholarship Program (DSS), granted by the Ministry of Presidential Affairs, UAE (M.A. PhD scholarship). This work was also partially funded by the Masdar Institute through the grant Novel Organic Optoelectronic Devices. The authors gratefully acknowledge Manfred Burghammer and Martin Rosenthal at the ID13 – the microfocus beamline at the ESRF for their assistance with the nXRD measurements. XMaS is a mid-range facility supported by the Engineering and Physical Sciences Research Council (EPSRC)