Capturing Peptide–GPCR Interactions and Their Dynamics

Many biological functions of peptides are mediated through G protein-coupled receptors (GPCRs). Upon ligand binding, GPCRs undergo conformational changes that facilitate the binding and activation of multiple effectors. GPCRs regulate nearly all physiological processes and are a favorite pharmacological target. In particular, drugs are sought after that elicit the recruitment of selected effectors only (biased ligands). Understanding how ligands bind to GPCRs and which conformational changes they induce is a fundamental step toward the development of more efficient and specific drugs. Moreover, it is emerging that the dynamic of the ligand–receptor interaction contributes to the specificity of both ligand recognition and effector recruitment, an aspect that is missing in structural snapshots from crystallography. We describe here biochemical and biophysical techniques to address ligand–receptor interactions in their structural and dynamic aspects, which include mutagenesis, crosslinking, spectroscopic techniques, and mass-spectrometry profiling. With a main focus on peptide receptors, we present methods to unveil the ligand–receptor contact interface and methods that address conformational changes both in the ligand and the GPCR. The presented studies highlight a wide structural heterogeneity among peptide receptors, reveal distinct structural changes occurring during ligand binding and a surprisingly high dynamics of the ligand–GPCR complexes

The Change in Attitudes Towards Abortion in Former West and East Germany After Reunification: a Latent Class Analysis and Implications for Abortion Access

Einleitung: Der rechtliche Status des Schwangerschaftsabbruchs in den Regionen der früheren DDR hat sich nach der Wiedervereinigung und der Übernahme der restriktiveren westdeutschen Politik geändert. Ziel dieser Studie war es, die Auswirkungen dieser Veränderungen auf die Einstellungen zum Schwangerschaftsabbruch und die Implikationen für die damit verbundene Gesundheitsversorgung in Ost- und Westdeutschland zu untersuchen. Material und Methoden: Der Allgemeinen Bevölkerungsumfrage der Sozialwissenschaften aus den Jahren 1992, 1996, 2000, 2006 und 2012 (N = 14 459) wurden bundesweit repräsentative Daten über das Ausmaß der öffentlichen Unterstützung für den legalen Zugang zum Schwangerschaftsabbruch entnommen. Basierend auf der Anzahl der vorhandenen Einrichtungen, die Schwangerschaftsabbrüche vornehmen, und dem Anteil der Frauen, die für einen Schwangerschaftsabbruch in ein anderes Bundesland reisten, wurden pro Bundesland 2 Indikatoren kalkuliert, welche die Einschränkungen beim Zugang zur Versorgung bei Schwangerschaftsabbrüchen darstellen sollten. Die Daten wurden mittels der latenten Klassenanalyse analysiert. Ergebnisse: Die Ergebnisse deuten darauf hin, dass die Einstellungen zum Schwangerschaftsabbruch in 3 unterschiedliche Untergruppen unterteilt werden können: 1) Der Zugang zum Schwangerschaftsabbruch wird, ungeachtet der Gründe der betroffenen Frauen, unterstützt; 2) Der Zugang zum Schwangerschaftsabbruch wird unterstützt, wenn der Schwangerschaftsabbruch mit einem mütterlichen oder fetalen Gesundheitsrisiko begründet wird, nicht aber, wenn er aus sozioökonomischen Gründen (z. B. finanzielle Einschränkungen) durchgeführt wird; und 3) der Zugang zum Schwangerschaftsabbruch wird generell nicht unterstützt. Die Größe der jeweiligen Untergruppen, die eine teilweise oder gänzliche Einschränkung des Zugangs zum Schwangerschaftsabbruchs befürworten, ist im Laufe des untersuchten Studienzeitraums in beiden Regionen angestiegen und diese Tendenz war nicht auf Veränderungen in den soziodemografischen Merkmale zurückzuführen. Befragte, die in Bundesländern lebten, wo der Zugang zur Versorgung bei Schwangerschaftsabbrüchen mit größeren Hürden verbunden war, neigten eher zu restriktiveren Einstellungen zum Schwangerschaftsabbruch. Schlussfolgerung: Die negativen Einstellungen zum Schwangerschaftsabbruch sind in West- und Ostdeutschland in den 2 Jahrzehnten seit der Wiedervereinigung angestiegen. Das kann sich auf Frauen nachteilig auswirken, wenn die allgemeine Akzeptanz der Versorgung und der Zugang zur Versorgung bei Schwangerschaftsabbrüchen sinkt. Politische Maßnahmen, der öffentliche Diskurs und die Integration von Schwangerschaftsabbrüchen in die Gesundheitsversorgung sollten sich nach den internationalen Richtlinien zum Schutz von Frauengesundheit und Frauenrechten richten.Introduction: The legal status of abortion has changed in the regions of former East Germany after reunification due to the adoption of restrictive West German abortion policies. The aim of this study was to evaluate the impact on attitudes towards abortion and the associated health care implications in Western and Eastern Germany. Materials and Methods: Nationally representative data on public support for legally restricting abortion access were taken from the German General Social Survey and included the surveys 1992, 1996, 2000, 2006 and 2012 (N = 14 459). Two indicators of barriers to access to abortion care were calculated for each federal state, based on the number of abortion facilities and the proportion of women seeking abortion outside their state of residency. Data were analysed using latent class analysis. Results: Results suggested that abortion attitudes could be classified into three distinct subgroups: 1) support for abortion access independent of womenʼs reason; 2) support on the basis of maternal or foetal health reasons but not for socio-economic reasons (e.g. financial restrictions); and 3) no support. The size of subgroups in favour of partial or complete restriction on abortion access increased in both regions over the study period and this trend could not be explained by changes in socio-demographic characteristics. Respondents living in a federal state with more barriers to access to abortion care were more likely to hold restrictive abortion attitudes. Conclusion: Negative attitudes towards abortion have increased in Western and Eastern Germany during the two decades following reunification and may harm women by limiting acceptability and accessibility of abortion care. Abortion policies, public discourse and provision of abortion care should be informed by international guidelines protecting womenʼs health and rights

A short guide to increase FAIRness of atmospheric model data

The generation, processing and analysis of atmospheric model data are expensive, as atmospheric model runs are often computationally intensive and the costs of ‘fast’ disk space are rising. Moreover, atmospheric models are mostly developed by groups of scientists over many years and therefore only few appropriate models exist for specific analyses, e.g. for urban climate. Hence, atmospheric model data should be made available for reuse by scientists, the public sector, companies and other stakeholders. Thereby, this leads to an increasing need for swift, user-friendly adaptation of standards.The FAIR data principles (Findable, Accessible, Interoperable, Reusable) were established to foster the reuse of data. Research data become findable and accessible if they are published in public repositories with general metadata and Persistent Identifiers (PIDs), e.g. DataCite DOIs. The use of PIDs should ensure that describing metadata is persistently available. Nevertheless, PIDs and basic metadata do not guarantee that the data are indeed interoperable and reusable without project-specific knowledge. Additionally, the lack of standardised machine-readable metadata reduces the FAIRness of data. Unfortunately, there are no common standards for non-climate models, e.g. for mesoscale models, available. This paper proposes a concept to improve the FAIRness of archived atmospheric model data. This concept was developed within the AtMoDat project (Atmospheric Model Data). The approach consists of several aspects, each of which is easy to implement: requirements for rich metadata with controlled vocabulary, the landing pages, file formats (netCDF) and the structure within the files. The landing pages are a core element of this concept as they should be human- and machine readable, hold discipline-specific metadata and present metadata on simulation and variable level. This guide is meant to help data producers and curators to prepare data for publication. Furthermore, this guide provides information for the choice of keywords, which supports data reusers in their search for data with search engines. © 2020 The author

The ATMODAT Standard enhances FAIRness of Atmospheric Model data

Within the AtMoDat project (Atmospheric Model Data, www.atmodat.de), a standard has been developed which is meant for improving the FAIRness of atmospheric model data published in repositories. Atmospheric model data form the basis to understand and predict natural events, including atmospheric circulation, local air quality patterns, and the planetary energy budget. Such data should be made available for evaluation and reuse by scientists, the public sector, and relevant stakeholders. Atmospheric modeling is ahead of other fields in many regards towards FAIR (Findable, Accessible, Interoperable, Reusable, see e.g. Wilkinson et al. (2016, doi:10.1101/418376)) data: many models write their output directly into netCDF or file formats that can be converted into netCDF. NetCDF is a non-proprietary, binary, and self-describing format, ensuring interoperability and facilitating reusability. Nevertheless, consistent human- and machine-readable standards for discipline-specific metadata are also necessary. While standardisation of file structure and metadata (e.g. the Climate and Forecast Conventions) is well established for some subdomains of the earth system modeling community (e.g. the Coupled Model Intercomparison Project, Juckes et al. (2020, https:doi.org/10.5194/gmd-13-201-2020)), other subdomains are still lacking such standardisation. For example, standardisation is not well advanced for obstacle-resolving atmospheric models (e.g. for urban-scale modeling). The ATMODAT standard, which will be presented here, includes concrete recommendations related to the maturity, publication, and enhanced FAIRness of atmospheric model data. The suggestions include requirements for rich metadata with controlled vocabularies, structured landing pages, file formats (netCDF), and the structure within files. Human- and machine-readable landing pages are a core element of this standard and should hold and present discipline-specific metadata on simulation and variable level

Warum und wie Sie Klimamodelldaten veröffentlichen sollten

Vorhersage und ProjektionBei Klimasimulationen werden große Mengen an Daten erzeugt. Aus diesem Grund können in der Regel nicht alle Ergebnisse einer Simulation eines Klimamodells von einer Forschungsgruppe alleine ausgewertet werden. Beim Coupled Model Intercomparison Projekt (CMIP) wird daher ein Fokus darauf gelegt, dass auch andere Forschungsgruppen die Daten auswerten können. Deshalb gibt es genaue Vorgaben, wie diese Daten zu beschreiben und zu formatieren sind. Zudem werden viele dieser Datensätze mit einem DOI (Digital Object Identifier) versehen. Dies alles erleichtert die Suche und Weiterverarbeitung der Daten. Allerdings gibt es weitaus mehr als CMIP Daten, die für die Klimaforschung wichtig sind. Viele Ergebnisse von z.B. regionalen Klimamodellen oder Stadtklimamodellen werden nicht veröffentlicht, obwohl von den Datenerzeugern nur ein Bruchteil der Ergebnisse ausgewertet werden kann. Deshalb drängen viele Förderer auf eine Veröffentlichung der Daten in einem Repositorium. Aber auch in diesem Fall können sie oft nicht weiterverwendet werden. Die Gründe sind vielfältig: Unzureichende Metadaten Mangelnde Auffindbarkeit, z.B. durch Suchmaschinen Fehlende Rechte zur Weiterverarbeitung Fehlende Qualitätskontrolle Das BMBF geförderte Projekt AtMoDat (https://www.ATMODAT.de) wurde 2019 gestartet, um die Veröffentlichung von Atmosphärischen Modelldaten zu stärken und zu verbessern. Eine Methode ist dabei die Einhaltung der FAIR-Prinzipien - Findable, Accessible, Interoperable, Reusable (siehe Wilkinson et al., 2016). Zudem sollten alle Daten mit einem DataCite DOI veröffentlicht werden, um die Auffindbarkeit und Zitierbarkeit zu verbessern. Eine Anleitung, wie man dabei vorgehen sollte, findet sich in dem Standard, der im AtMoDat-Projekt entwickelt wurde. Der ATMODAT-Standard ist leicht umzusetzen und beinhaltet genaue Vorgaben für die Metadaten des DOI, die Landing Page und die Header der netCDF-Dateien. Daten, die diesem Standard genügen und dessen Einhaltung vom jeweiligen Repositorium geprüft wurde, können mit dem Earth System Data Branding (EASYDAB) versehen werden. Durch dieses Branding kann eine angemessene Qualitätssicherung der Daten durch die Nutzer sehr leicht nachvollzogen werden. Im Vortrag werden der Standard und EASYDAB vorgestellt

Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements

The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron–electron resonance (DEER) pulsed EPR technique on the receptor

Cell-Free Expression and Photo-Crosslinking of the Human Neuropeptide Y2 Receptor

G protein-coupled receptors (GPCRs) represent a large family of different proteins, which are involved in physiological processes throughout the entire body. Furthermore, they represent important drug targets. For rational drug design, it is important to get further insights into the binding mode of endogenous ligands as well as of therapeutic agents at the respective target receptors. However, structural investigations usually require homogenous, solubilized and functional receptors, which is still challenging. Cell-free expression methods have emerged in the last years and many different proteins are successfully expressed, including hydrophobic membrane proteins like GPCRs. In this work, an Escherichia coli based cell-free expression system was used to express the neuropeptide Y2 receptor (Y2R) for structural investigations. This GPCR was expressed in two different variants, a C-terminal enhanced green fluorescent fusion protein and a cysteine deficient variant. In order to obtain soluble receptors, the expression was performed in the presence of mild detergents, either Brij-35 or Brij-58, which led to high amounts of soluble receptor. Furthermore, the influence of temperature, pH value and additives on protein expression and solubilization was tested. For functional and structural investigations, the receptors were expressed at 37°C, pH 7.4 in the presence of 1 mM oxidized and 5 mM reduced glutathione. The expressed receptors were purified by ligand affinity chromatography and functionality of Y2R_cysteine_deficient was verified by a homogenous binding assay. Finally, photo-crosslinking studies were performed between cell-free expressed Y2R_cysteine_deficient and a neuropeptide Y (NPY) analog bearing the photoactive, unnatural amino acid p-benzoyl-phenylalanine at position 27 and biotin at position 22 for purification. After enzymatic digestion, fragments of crosslinked receptor were identified by mass spectrometry. Our findings demonstrate that, in contrast to Y1R, NPY position 27 remains flexible when bound to Y2R. These results are in agreement with the suggested binding mode of NPY at Y2R

The Dynamics of the Neuropeptide Y Receptor Type 1 Investigated by Solid-State NMR and Molecular Dynamics Simulation

We report data on the structural dynamics of the neuropeptide Y (NPY) G-protein-coupled receptor (GPCR) type 1 (Y1R), a typical representative of class A peptide ligand GPCRs, using a combination of solid-state NMR and molecular dynamics (MD) simulation. First, the equilibrium dynamics of Y1R were studied using 15N-NMR and quantitative determination of 1H-13C order parameters through the measurement of dipolar couplings in separated-local-field NMR experiments. Order parameters reporting the amplitudes of the molecular motions of the C-H bond vectors of Y1R in DMPC membranes are 0.57 for the Cα sites and lower in the side chains (0.37 for the CH2 and 0.18 for the CH3 groups). Different NMR excitation schemes identify relatively rigid and also dynamic segments of the molecule. In monounsaturated membranes composed of longer lipid chains, Y1R is more rigid, attributed to a higher hydrophobic thickness of the lipid membrane. The presence of an antagonist or NPY has little influence on the amplitude of motions, whereas the addition of agonist and arrestin led to a pronounced rigidization. To investigate Y1R dynamics with site resolution, we conducted extensive all-atom MD simulations of the apo and antagonist-bound state. In each state, three replicas with a length of 20 μs (with one exception, where the trajectory length was 10 μs) were conducted. In these simulations, order parameters of each residue were determined and showed high values in the transmembrane helices, whereas the loops and termini exhibit much lower order. The extracellular helix segments undergo larger amplitude motions than their intracellular counterparts, whereas the opposite is observed for the loops, Helix 8, and termini. Only minor differences in order were observed between the apo and antagonist-bound state, whereas the time scale of the motions is shorter for the apo state. Although these relatively fast motions occurring with correlation times of ns up to a few µs have no direct relevance for receptor activation, it is believed that they represent the prerequisite for larger conformational transitions in proteins

ATMODAT Standard v3.0

Within the AtMoDat project (Atmospheric Model Data), a standard has been developed which is meant for improving the FAIRness of atmospheric model data published in repositories. The ATMODAT standard includes concrete recommendations related to the maturity, publication and enhanced FAIRness of atmospheric model data. The suggestions include requirements for rich metadata with controlled vocabularies, structured landing pages, file formats (netCDF) and the structure within files. Human- and machine readable landing pages are a core element of this standard, and should hold and present discipline-specific metadata on simulation and variable level. This standard is an updated and translated version of "Bericht über initialen Kernstandard und Kurationskriterien des AtMoDat Projektes (v2.4