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  1. Article: Spectroscopically Orthogonal Labelling to Disentangle Site-Specific Nitroxide Label Distributions.

    Vitali, Valentina / Ackermann, Katrin / Hagelueken, Gregor / Bode, Bela E

    Applied magnetic resonance

    2023  Volume 55, Issue 1-3, Page(s) 187–205

    Abstract: Biomolecular applications of pulse dipolar electron paramagnetic resonance spectroscopy (PDS) are becoming increasingly valuable in structural biology. Site-directed spin labelling of proteins is routinely performed using nitroxides, with paramagnetic ... ...

    Abstract Biomolecular applications of pulse dipolar electron paramagnetic resonance spectroscopy (PDS) are becoming increasingly valuable in structural biology. Site-directed spin labelling of proteins is routinely performed using nitroxides, with paramagnetic metal ions and other organic radicals gaining popularity as alternative spin centres. Spectroscopically orthogonal spin labelling using different types of labels potentially increases the information content available from a single sample. When analysing experimental distance distributions between two nitroxide spin labels, the site-specific rotamer information has been projected into the distance and is not readily available, and the contributions of individual labelling sites to the width of the distance distribution are not obvious from the PDS data. Here, we exploit the exquisite precision of labelling double-histidine (dHis) motifs with Cu
    Supplementary information: The online version contains supplementary material available at 10.1007/s00723-023-01611-1.
    Language English
    Publishing date 2023-09-24
    Publishing country Austria
    Document type Journal Article
    ZDB-ID 1480644-7
    ISSN 1613-7507 ; 0937-9347
    ISSN (online) 1613-7507
    ISSN 0937-9347
    DOI 10.1007/s00723-023-01611-1
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  2. Book ; Online ; Thesis: Integrated structural biology investigation of the bacterial TRAP transporter SiaPQM from pathogenic bacteria

    Glänzer, Janin [Verfasser] / Hagelüken, Gregor [Akademischer Betreuer] / Kubitschek, Ulrich [Gutachter]

    2023  

    Author's details Janin Glänzer ; Gutachter: Ulrich Kubitschek ; Betreuer: Gregor Hagelüken
    Keywords Biowissenschaften, Biologie ; Life Science, Biology
    Subject code sg570
    Language English
    Publisher Universitäts- und Landesbibliothek Bonn
    Publishing place Bonn
    Document type Book ; Online ; Thesis
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  3. Article ; Online: Conformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaP.

    Peter, Martin F / Ruland, Jan A / Kim, Yeojin / Hendricks, Philipp / Schneberger, Niels / Siebrasse, Jan Peter / Thomas, Gavin H / Kubitscheck, Ulrich / Hagelueken, Gregor

    Nature communications

    2024  Volume 15, Issue 1, Page(s) 217

    Abstract: The tripartite ATP-independent periplasmic (TRAP) transporters use an extra cytoplasmic substrate binding protein (SBP) to transport a wide variety of substrates in bacteria and archaea. The SBP can adopt an open- or closed state depending on the ... ...

    Abstract The tripartite ATP-independent periplasmic (TRAP) transporters use an extra cytoplasmic substrate binding protein (SBP) to transport a wide variety of substrates in bacteria and archaea. The SBP can adopt an open- or closed state depending on the presence of substrate. The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient. Insights from experimental structures, structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein. Here, we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM from Haemophilus influenzae in different conformational states. The SBP, HiSiaP, is locked in its substrate-bound form and the transmembrane elevator, HiSiaQM, is locked in either its assumed inward- or outward-facing states. We characterize the disulfide-locked constructs and use single-molecule total internal reflection fluorescence (TIRF) microscopy to study their interactions. Our experiments demonstrate that the SBP and the transmembrane elevator are indeed conformationally coupled, meaning that the open and closed state of the SBP recognize specific conformational states of the transporter and vice versa.
    MeSH term(s) Carrier Proteins ; N-Acetylneuraminic Acid ; Membrane Transport Proteins/genetics ; Molecular Conformation ; Disulfides
    Chemical Substances Carrier Proteins ; N-Acetylneuraminic Acid (GZP2782OP0) ; Membrane Transport Proteins ; Disulfides
    Language English
    Publishing date 2024-01-08
    Publishing country England
    Document type Journal Article
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-023-44327-3
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  4. Article ; Online: PELDOR/DEER: An Electron Paramagnetic Resonance Method to Study Membrane Proteins in Lipid Bilayers.

    Peter, Martin F / Bountra, Kiran / Beis, Konstantinos / Hagelueken, Gregor

    Methods in molecular biology (Clifton, N.J.)

    2021  Volume 2168, Page(s) 313–333

    Abstract: Every membrane protein is involved in close interactions with the lipid environment of cellular membranes. The annular lipids, that are in direct contact with the polypeptide, can in principle be seen as an integral part of its structure, akin to the ... ...

    Abstract Every membrane protein is involved in close interactions with the lipid environment of cellular membranes. The annular lipids, that are in direct contact with the polypeptide, can in principle be seen as an integral part of its structure, akin to the first hydration shell of soluble proteins. It is therefore desirable to investigate the structure of membrane proteins and especially their conformational flexibility under conditions that are as close as possible to their native state. This can be achieved by reconstituting the protein into proteoliposomes, nanodiscs, or bicelles. In recent years, PELDOR/DEER spectroscopy has proved to be a very useful method to study the structure and function of membrane proteins in such artificial membrane environments. The technique complements both X-ray crystallography and cryo-EM and can be used in combination with virtually any artificial membrane environment and under certain circumstances even in native membranes. Of the above-mentioned membrane mimics, bicelles are currently the least often used for PELDOR studies, although they offer some advantages, especially their ease of use. Here, we provide a step-by-step protocol for studying a bicelle reconstituted membrane protein with PELDOR/DEER spectroscopy.
    MeSH term(s) ATP-Binding Cassette Transporters/chemistry ; ATP-Binding Cassette Transporters/metabolism ; Cell Membrane/metabolism ; Electron Spin Resonance Spectroscopy/methods ; Escherichia coli/metabolism ; Escherichia coli Proteins/chemistry ; Escherichia coli Proteins/metabolism ; Lipid Bilayers/chemistry ; Lipid Bilayers/metabolism ; Molecular Conformation ; Spin Labels
    Chemical Substances ATP-Binding Cassette Transporters ; Escherichia coli Proteins ; Lipid Bilayers ; McjD protein, E coli ; Spin Labels
    Language English
    Publishing date 2021-02-13
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-0716-0724-4_15
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  5. Article ; Online: Pyroptosis inhibiting nanobodies block Gasdermin D pore formation.

    Kopp, Anja / Hagelueken, Gregor / Jamitzky, Isabell / Moecking, Jonas / Schiffelers, Lisa D J / Schmidt, Florian I / Geyer, Matthias

    Nature communications

    2023  Volume 14, Issue 1, Page(s) 7923

    Abstract: Human Gasdermin D (GSDMD) is a key mediator of pyroptosis, a pro-inflammatory form of cell death occurring downstream of inflammasome activation as part of the innate immune defence. Upon cleavage by inflammatory caspases in the cytosol, the N-terminal ... ...

    Abstract Human Gasdermin D (GSDMD) is a key mediator of pyroptosis, a pro-inflammatory form of cell death occurring downstream of inflammasome activation as part of the innate immune defence. Upon cleavage by inflammatory caspases in the cytosol, the N-terminal domain of GSDMD forms pores in the plasma membrane resulting in cytokine release and eventually cell death. Targeting GSDMD is an attractive way to dampen inflammation. In this study, six GSDMD targeting nanobodies are characterized in terms of their binding affinity, stability, and effect on GSDMD pore formation. Three of the nanobodies inhibit GSDMD pore formation in a liposome leakage assay, although caspase cleavage was not perturbed. We determine the crystal structure of human GSDMD in complex with two nanobodies at 1.9 Å resolution, providing detailed insights into the GSDMD-nanobody interactions and epitope binding. The pore formation is sterically blocked by one of the nanobodies that binds to the oligomerization interface of the N-terminal domain in the multi-subunit pore assembly. Our biochemical and structural findings provide tools for studying inflammasome biology and build a framework for the design of GSDMD targeting drugs.
    MeSH term(s) Humans ; Caspases/metabolism ; Gasdermins ; Inflammasomes/metabolism ; Intracellular Signaling Peptides and Proteins/metabolism ; Phosphate-Binding Proteins/metabolism ; Pyroptosis ; Single-Domain Antibodies/metabolism
    Chemical Substances Caspases (EC 3.4.22.-) ; Gasdermins ; Inflammasomes ; Intracellular Signaling Peptides and Proteins ; Phosphate-Binding Proteins ; Single-Domain Antibodies
    Language English
    Publishing date 2023-12-01
    Publishing country England
    Document type Journal Article
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-023-43707-z
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  6. Article ; Online: Adenylation Domain-Guided Recruitment of

    Wirtz, Daniel A / Schneberger, Niels / Klöppel, Sophie / Richarz, René / Geyer, Matthias / König, Gabriele M / Hagelueken, Gregor / Crüsemann, Max

    ACS chemical biology

    2023  Volume 18, Issue 8, Page(s) 1748–1759

    Abstract: Nonheme diiron monooxygenases (NHDMs) interact with nonribosomal peptide synthetase (NRPS) assembly lines to install β-hydroxylations at thiolation-domain-bound amino acids during nonribosomal peptide biosynthesis. The high potential of this enzyme ... ...

    Abstract Nonheme diiron monooxygenases (NHDMs) interact with nonribosomal peptide synthetase (NRPS) assembly lines to install β-hydroxylations at thiolation-domain-bound amino acids during nonribosomal peptide biosynthesis. The high potential of this enzyme family to diversify the products of engineered assembly lines is disproportionate to the currently small knowledge about their structures and mechanisms of substrate recognition. Here, we report the crystal structure of FrsH, the NHDM which catalyzes the β-hydroxylation of l-leucines during biosynthesis of the depsipeptide G protein inhibitor FR900359. Using biophysical approaches, we provide evidence that FrsH interacts with the cognate monomodular NRPS FrsA. By AlphaFold modeling and mutational studies, we detect and examine structural features within the assembly line crucial to recruit FrsH for leucine β-hydroxylation. These are, in contrast to cytochrome-dependent NRPS β-hydroxylases, not located on the thiolation domain, but on the adenylation domain. FrsH can be functionally substituted by homologous enzymes from biosyntheses of the cell-wall-targeting antibiotics lysobactin and hypeptin, indicating that these features are generally applicable to members of the family of
    MeSH term(s) Mixed Function Oxygenases/metabolism ; Peptide Biosynthesis, Nucleic Acid-Independent ; Amino Acids/chemistry ; Anti-Bacterial Agents ; Peptide Synthases/metabolism
    Chemical Substances Mixed Function Oxygenases (EC 1.-) ; Amino Acids ; Anti-Bacterial Agents ; Peptide Synthases (EC 6.3.2.-)
    Language English
    Publishing date 2023-06-27
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1554-8937
    ISSN (online) 1554-8937
    DOI 10.1021/acschembio.3c00106
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  7. Article ; Online: Studying structure and function of membrane proteins with PELDOR/DEER spectroscopy - The crystallographers' perspective.

    Glaenzer, Janin / Peter, Martin F / Hagelueken, Gregor

    Methods (San Diego, Calif.)

    2018  Volume 147, Page(s) 163–175

    Abstract: In 1985, the first X-ray structure of a membrane protein was determined. Today, more than 30 years later, many more structures have been solved. Nevertheless, studying the structure of membrane proteins remains a very challenging task. Due to their ... ...

    Abstract In 1985, the first X-ray structure of a membrane protein was determined. Today, more than 30 years later, many more structures have been solved. Nevertheless, studying the structure of membrane proteins remains a very challenging task. Due to their inherent conformational flexibility, having a single X-ray structure is usually only the first step towards truly understanding the function of these dynamic molecules. For this reason, additional methods are needed that can provide complementary information, especially about conformational flexibility. Pulsed electron-electron double resonance spectroscopy (PELDOR, also known as DEER) is such a method. It can be used to precisely measure nanometer distance distributions between intrinsic or artificially introduced spin-centers in macromolecules and thereby to probe the conformational state of the macromolecule. PELDOR can be applied in solution, in detergent, in lipid bilayers and even within cells. However, PELDOR is an advanced spectroscopy technique and requires specialised equipment and training. This chapter aims to be a starting point for crystallographers and other structural biologists who want to get a better understanding of PELDOR spectroscopy and its application. It gives an insight into the planning stages of the experiment (i.e., which spin labels are possible and where to place them), how a PELDOR experiment is conducted and how the results are interpreted. For this purpose, the substrate binding protein (SBP) from a Vibrio cholerae TRAP transporter is used as a step-by-step example. Further, the chapter gives examples of how PELDOR spectroscopy has previously been applied to overcome known limitations of X-ray crystallography in modern integrative structural biology approaches.
    MeSH term(s) Crystallography ; Electron Spin Resonance Spectroscopy/methods ; Fluorescence Resonance Energy Transfer ; Membrane Proteins/chemistry ; Membrane Proteins/physiology ; Protein Conformation
    Chemical Substances Membrane Proteins
    Language English
    Publishing date 2018-03-03
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1066584-5
    ISSN 1095-9130 ; 1046-2023
    ISSN (online) 1095-9130
    ISSN 1046-2023
    DOI 10.1016/j.ymeth.2018.03.002
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  8. Book ; Online ; Thesis: Structural and functional characterization of TRAP transporters from bacterial pathogens

    Peter, Martin Friedrich [Verfasser] / Hagelüken, Gregor [Akademischer Betreuer] / Kubitscheck, Ulrich [Gutachter]

    2021  

    Author's details Martin Friedrich Peter ; Gutachter: Ulrich Kubitscheck ; Betreuer: Gregor Hagelüken
    Keywords Naturwissenschaften ; Science
    Subject code sg500
    Language English
    Publisher Universitäts- und Landesbibliothek Bonn
    Publishing place Bonn
    Document type Book ; Online ; Thesis
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  9. Article ; Online: Directionality of PYD filament growth determined by the transition of NLRP3 nucleation seeds to ASC elongation.

    Hochheiser, Inga V / Behrmann, Heide / Hagelueken, Gregor / Rodríguez-Alcázar, Juan F / Kopp, Anja / Latz, Eicke / Behrmann, Elmar / Geyer, Matthias

    Science advances

    2022  Volume 8, Issue 19, Page(s) eabn7583

    Abstract: Inflammasomes sense intrinsic and extrinsic danger signals to trigger inflammatory responses and pyroptotic cell death. Homotypic pyrin domain (PYD) interactions of inflammasome forming nucleotide-binding oligomerization domain (NOD)-like receptors with ... ...

    Abstract Inflammasomes sense intrinsic and extrinsic danger signals to trigger inflammatory responses and pyroptotic cell death. Homotypic pyrin domain (PYD) interactions of inflammasome forming nucleotide-binding oligomerization domain (NOD)-like receptors with the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) mediate oligomerization into filamentous assemblies. We describe the cryo-electron microscopy (cryo-EM) structure of the human NLRP3
    Language English
    Publishing date 2022-05-13
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2810933-8
    ISSN 2375-2548 ; 2375-2548
    ISSN (online) 2375-2548
    ISSN 2375-2548
    DOI 10.1126/sciadv.abn7583
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  10. Article ; Online: Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET.

    Peter, Martin F / Gebhardt, Christian / Mächtel, Rebecca / Muñoz, Gabriel G Moya / Glaenzer, Janin / Narducci, Alessandra / Thomas, Gavin H / Cordes, Thorben / Hagelueken, Gregor

    Nature communications

    2022  Volume 13, Issue 1, Page(s) 4396

    Abstract: Pulsed electron-electron double resonance spectroscopy (PELDOR/DEER) and single-molecule Förster resonance energy transfer spectroscopy (smFRET) are frequently used to determine conformational changes, structural heterogeneity, and inter probe distances ... ...

    Abstract Pulsed electron-electron double resonance spectroscopy (PELDOR/DEER) and single-molecule Förster resonance energy transfer spectroscopy (smFRET) are frequently used to determine conformational changes, structural heterogeneity, and inter probe distances in biological macromolecules. They provide qualitative information that facilitates mechanistic understanding of biochemical processes and quantitative data for structural modelling. To provide a comprehensive comparison of the accuracy of PELDOR/DEER and smFRET, we use a library of double cysteine variants of four proteins that undergo large-scale conformational changes upon ligand binding. With either method, we use established standard experimental protocols and data analysis routines to determine inter-probe distances in the presence and absence of ligands. The results are compared to distance predictions from structural models. Despite an overall satisfying and similar distance accuracy, some inconsistencies are identified, which we attribute to the use of cryoprotectants for PELDOR/DEER and label-protein interactions for smFRET. This large-scale cross-validation of PELDOR/DEER and smFRET highlights the strengths, weaknesses, and synergies of these two important and complementary tools in integrative structural biology.
    MeSH term(s) Cysteine/chemistry ; Electron Spin Resonance Spectroscopy/methods ; Fluorescence Resonance Energy Transfer/methods ; Ligands ; Proteins ; Spin Labels
    Chemical Substances Ligands ; Proteins ; Spin Labels ; Cysteine (K848JZ4886)
    Language English
    Publishing date 2022-07-29
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-022-31945-6
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