GPF Symposium 2023

Europe/Berlin
MPI-NAT City Campus

MPI-NAT City Campus

    • 10:00 AM 11:40 AM
      Session 1 : Proximity labeling and mass spectrometry of native protein complexes
      • 10:00 AM
        Oliver Valerius – Georg-August-Universität Göttingen 20m

        Analysis of protein proximities with biotin labeling and mass spectrometry (BioID) – LCMS core facility support for BioID

        Together with the Göttingen Proteomics Forum (GPF) our university proteomics core facility applied for and was granted DFG funding to establish support and service capacity for research groups that intend to start and conduct (split-)BioID experiments in cooperation (GoeCoOp-PL/MS). Engineered biotin ligases fused to bait proteins enable in vivo proximity labeling of transiently colocalizing proteins with biotin followed by protein-biotin affinity capture and LCMS analysis. Dynamic protein remodeling within specific cellular microenvironments accounts for diverse regulatory and structural processes. In our lab, we established quantitative (split-)BioID to study transient colocalization of proteins within the head region of the small 40S ribosomal subunit (hr40S) in yeast. We study the function and signaling impact of the Gbeta-like protein RACK1 that integrally resides at the hr40S and significantly affects the ribosomal quality control (RQC) and signal transduction pathways.

      • 10:20 AM
        Julien Béthune – Hochschule für Angewandte Wissenschaften Hamburg 40m

        UltraID, a small-size hyperactive enzyme for proximity-dependent biotinylation

        Proximity-dependent biotinylation (PDB) has become a standard technique in the toolbox of methods available for the identification of protein-protein interactions. The most commonly used approach is based on the original BioID technique in which an abortive variant of a prokaryotic biotin protein ligase is fused to a protein of interest to mediate the non-specific biotinylation of vicinal proteins in living cells. While the first generation of PDB enzymes led to successful identification of protein-protein interactions, they also came with drawbacks, mostly bulky size and/or slow labeling kinetics. This triggered efforts to develop novel enzymes with improved properties to render the assay more versatile. Recently we engineered ultraID the smallest size and most efficient enzyme for PDB described to date. With ultraID we probed the membrane-associated interactome of the COPI coat protein complex and thereby demonstrated the power of PDB to access protein-protein interactions that transiently happen on a specific subcellular organelle.

      • 11:00 AM
        Nina Morgner – Goethe-Universität Frankfurt 40m

        Native mass spectrometry: How to probe molecular principles of assembly and interactions of protein complexes

        Nina Morgner, Rene Zangl, Jonathan Schulte

        Protein complex assembly as well as their interplay are controlled by the non-covalent interactions of all biomolecular partners. Native mass spectrometry and ion mobility are ideally suited to unravel the molecular principles which tightly control these interactions. Here I will present what we can learn about well-choreographed assembly strategies of a multi protein complex such as an ATPase or rather unwanted aggregation as seen for the Alzheimer related Amyloid beta peptide. For the example of photoreceptors I will show how instrumental modifications can allow for time resolved studies of light dependent conformational rearrangements upon illumination.

    • 11:40 AM 12:40 PM
      Lunch break 1h
    • 12:40 PM 2:20 PM
      Session 2 : Crosslinking
      • 12:40 PM
        Henning Urlaub – MPI für Multidisziplinäre Naturwissenschaften, Göttingen 40m

        RNA/DNA-protein crosslinking mass spectrometry - Applications of UV and chemical crosslinking

        Crosslinking mass spectrometry (XL-MS) can resolve sites of interaction in protein-RNA and DNA complexes at single amino acid and nucleic acid resolution. Processing of (large) XL-MS data, reliable assignment, and visualization of crosslinking results – including annotation of spectra –remain key challenges in the database search of crosslinked species. We have developed NuXL, a database search engine, which robustly identifies UV and chemically induced crosslink sites within peptide sequences from MS2 spectra, and significantly improves data-processing and identification of protein–RNA/DNA crosslinks, irrespective of source (i.e., isolated complexes or cellular entities).

      • 1:20 PM
        Andrew Norris – Thermo Fisher Scientific 20m

        Bound Together: The picture-perfect marriage of crosslinking mass spectrometry and Cryo-EM

        Yuqi Shi(1); Ye Hi(1); Leigh Foster(2), Albert Konijnenberg(3), Ryan Bomgarden(2), Rosa Viner(1)

        Thermo Fisher Scientific, (1) San Jose, CA, (2) Rockford, IL, (3) Eindhoven, NL

        Cross-linking mass spectrometry (XL-MS) has become a powerful structural tool for mapping protein-protein interactions and providing distance constraints that help elucidate architectures of large protein complexes. These interaction identities and constraints complement existing cryo-EM structures and make XL-MS one of the most used mass spectrometry techniques in conjunction with Single Particle Analysis (SPA). In comparison to other structural methods, XL-MS approaches offer distinct advantages due to the speed, accuracy, sensitivity, and versatility of the method, especially for the study of heterogeneous and dynamic protein complexes. In this presentation, we will introduce the development of an easy to use, complete XL-MS workflow starting from sample prep to data analysis and incorporating results into an integrative modeling platform via Chimera tools. In addition, examples of the use of XL-MS in characterizing protein-nucleic acid interactions and structural dynamics of protein complexes for both SPA and cryo-tomography applications will be presented.

      • 1:40 PM
        Juri Rappsilber – Technische Universität Berlin 40m

        Protein structures inside cells – how and why

        Accurately modelling the structures of proteins and their complexes using artificial intelligence is revolutionizing molecular biology. Remaining challenges result from the limited information available on specific protein states including protein-protein interactions. We present how experimental data enable systematically modelling novel protein assemblies and how experimental data can inject information about in-cell states of proteins into the modelling workflow. We report and validate novel interactors of central cellular machineries that include the ribosome, RNA polymerase, and pyruvate dehydrogenase, assigning function to several uncharacterized proteins in the model organism Bacillus subtilis. Our approach uncovers protein-protein interactions inside intact cells, provides structural insight into their interaction interfaces, and is applicable to genetically intractable organisms, including pathogenic bacteria. Facile generation of pseudo-atomic models of PPIs provides a plausible shortcut to the improved functional understanding of the many currently uncharacterised proteins.

    • 2:20 PM 2:50 PM
      Coffee break 30m
    • 2:50 PM 4:15 PM
      Session 3: Biotin Identification (BioID)
      • 2:50 PM
        Ralf-Peter Jansen – Eberhard Karls Universität Tübingen 40m

        (RNA-)BioID as tool to probe for RNA-protein association

      • 3:30 PM
        Noa Lipstein – Leibniz Forschungsinstitut für Molekulare Pharmakologie, Berlin 40m

        A Proximity Map of the Presynaptic Terminal

        In the brain, information is transferred between neurons at synaptic connections. Synapses show a high degree of structural and functional diversity, and undergo continuous adaptation during development, in response to stimuli, and in disease. To discover molecular mechanisms that underlie synaptic diversity and plasticity, we established proximity labeling approaches coupled to mass spectrometric analysis in vitro and in vivo in the mouse brain. We generated a proximity map of the presynaptic compartment, and propose that the nano-organization of signaling complexes is a major generator of synaptic heterogeneity and diversity.

      • 4:10 PM
        Closing remarks 5m