P01 Insights into lipolysaccharide (LPS) transport by solid-state NMR spectroscopy
PI: Clemens Glaubitz
Lipopolysaccharides (LPS) are the main component of the asymmetric outer membrane of Gram-negative bacteria and protect them against antibiotics and the mammalian immune system. One essential step in maintaining this asymmetry is the translocation of core-LPS across the inner membrane by the ABC floppase MsbA from where LPS is carried on towards the outer membrane by the lipopolysaccharide transport (LPT) system. In project P01, we will address the LPS transport mechanism and explore how its presence alters membrane properties by solid-state NMR.
P02 Mechanism of bacterial transenvelope protein and lipid transport
PI: Benesh Joseph
The cell envelope of Gram-negative bacteria consists of an inner membrane (IM) and an outer membrane (OM) that protects cells from harsh conditions. The OM is an asymmetric bilayer made up of phosphlipids (PL) and lipopolysaccharides (LPS) and harbors numerous ß-barrel proteins (outer membrane proteins, OMPs). The LptDE translocon mediates the final step of LPS insertion into the OM. Folding and insertion of unfolded OMPs from the periplasm into the OM is mediated by the ß-barrel assembly machinery (BAM) with the assistance of periplasmic chaperones, primarily SurA. We will use in situ pulsed electron spin resonance (ESR) spectroscopy combined with other biophysical approaches to elucidate the structural and dynamical basis for outer membrane biogenesis. For SurA, its substrate-dependent conformational rearrangements and interaction with BAM complex will be investigated. For LptDE, conformational dynamics/heterogeneity of its key structural elements will be characterized in the native membrane environment in presence of LPS or the periplasmic LPS binding protein LptA. The binding and inhibition mode for the peptidomimetic antibiotic thanatin on LptDE will be investigated in a similar manner. Finally, we will develop new ESR approaches to study heterooligomeric OMP complexes in the native membranes employing different spin labels and unnatural amino acids.
P03 Molecular basis of lipid transport across the periplasm
The periplasm of Gram-negative bacteria is confined by the inner membrane (IM) composed of a bilayer of phospholipids and the outer membrane (OM), with an inner layer of phospholipids and an outer layer of mainly lipopolysaccharides. The preservation of the OM-lipid asymmetry is necessary to protect the cell from hydrophobic toxins and is in Campylobacter jejuni thought to based on the transport of phospholipids between the IM and OM by an RND transport machinery and MlaA/MlaC homologs. In project P03, we will investigate the role of the putative lipid RND transporter, its dependence on MlaA and MlaC homologs, its structure, function, and the molecular mechanism of lipid transport through the periplasm.
P04 Regulation of potassium channel YugO in bacterial electrical signaling
PI: Inga Hänelt
In biofilms, bacteria use electrical signaling for cell-cell communication. In Bacillus subtilis, potassium channel YugO is suggested to be a key player by mediating potassium efflux at nitrogen starvation. In project P04, we will elucidate the molecular principles of electrical signaling by structurally and functionally characterizing YugO and identifying the network required for activation.
P05 Molecular dynamics of light responsive molecular machines
PI: Josef Wachtveitl
In project P05, the conformational dynamics of microbial rhodopsnins will be studied using time-resolved UV/vis and IR spectroscopy manipulated by multipulse techniques. These optical control patterns will be applied to protein assemblies and networks. We also aim to establish optogenetically relevant carotenoid-based voltage indicators and to monitor their function using spectroscopic and imaging techniques.
P06 Optogenetic analysis of membrane compartmentalization between and within excitable cells
Networks of excitable cells are formed by electrical (gap) junctions. In project P06, we will address the role of gap junction subunits (and accessory stomatins) in muscular and nervous systems of an intact animal (Caenorhabditis elegans), using state-of-the-art optogenetic actuation and voltage imaging in closed-loop. This project will decipher such gap junction networks acting in muscular electrical compartmentalization, and in neural circuit function regulating behavior.
P07 Dynamics of membrane receptor clustering in endothelial cells
The spatial organization of transmembrane receptors is a critical step in propagation of signals and receptor trafficking in cells. In project P07, we will specifically characterize the dynamic spatial arrangement of ephrinB homotypic and heterotypic complexes with its partners by super-resolution microscopy. We will also investigate how these arrangements are regulated at the membrane, recruiting cytosolic adaptors to achieve a variety of efficient functional outcomes.
P08 Quantitative super-resolution microscopy unravels nanoscale patterns of membrane receptor networks
PI: Mike Heilemann
Project P08 studies the composition and dynamics of receptor-ligand assemblies in their native cellular environment and will answer quesions about structure-function relationships, ligand-driven selective activation, and protein-protein communication. To this end, we will employ super-resolution optical microscopy methods capable of visualizing multiple proteins and reporting on stoichiometries and spatial organization, as well as single-molecule imaging of protein assembly kinetics in living cells.
P09 Artificial intelligence-guided molecular dynamics simulations decipher the activation mechanism of cellular control programs
PI: Roberto Covino
Project P09 will use modeling and simulations to gain a mechanistic understanding of fundamental regulatory processes. We will investigate the activation of the unfolded protein response (UPR), a pathway central in health and disease. We will establish artificial intelligence-guided simulations to understand how membrane-mediated interactions control the activation of the UPR. Furthermore, we will develop an interdisciplinary strategy to unravel the dimerization and activation of MET receptors.
P10 Modulation of tyrosine receptor signaling investigated by NMR spectroscopy
PI: Harald Schwalbe
The central aim of project P10 is to investigate fundamental aspects of tyrosine kinase receptor signaling pathways for two pharmacological relevant receptors (FGFR and EPH) and their respective ligands. The dynamics of receptor/ligand interaction and the influence of inhibitors of low molecular weight will be investigated, using several biophysical and structural biology techniques, in particular NMR spectroscopy, available within CRC 1507.
P11 Structure and dynamics of the TRPV4 ion channel as a cellular signaling hub
PI: Ute A. Hellmich
Project P11 focuses on transient receptor potential vanilloid 4 (TRPV4) as the mediator of multipartite and highly dynamic interactions with lipid and protein partners in the cell. TRPV4 structural dynamics, the interaction of the N-terminus, and its large intrinsically disordered region with the membrane, Pacsin proteins and the cytoskeleton as well as the molecular consequences of disease mutants will be investigated.
P12 Molecular dynamics simulations of membrane perforation machineries and the nuclear pore complex
PI: Gerhard Hummer
Project P12 uses molecular dynamics simulations to study key steps in the assembly and function of two archetypal membrane pores in eukaryotes: (i) the nuclear pore complex governing the transport between the nucleus and the cytosol, and (ii) the ring-shaped cytolysin-like pores formed by gasdermin in inflammation.
P13 Probing multiprotein assemblies with native mass spectrometry
PI: Nina Morgner
Mitochondrial ATPases are of high relevance for (human) life. In project P13, we will probe changes in the ATP synthase complex formation in correlation to mutations causing neuropathological diseases by native mass spectrometry. In addition, we want to investigate different mechanistic principles, which control recruitment of specific phospholipids or lipopolysaccharides. To advance these aims, we will push boundaries regarding the use of membrane mimics for native mass spectrometry.
P14 Structural basis of complex I function and assembly
Respiratory complex I is a 1MDa membrane protein complex with a central role in energy metabolism. In humans, assembly of the intricate molecular machine (44 subunits, 9 redox active co-factors) requires more than 15 assembly factors. We use complex I from the aerobic yeast Yarrowia lipolytica as a model system. Our high-resolution cryo-EM structures of complex I in native form and during steady state activity have enabled us to define proton translocation pathways by protein-bound water molecules and to monitor detailed conformational changes during turnover. Our first goal is to gain a comprehensive understanding of the catalytic mechanism of complex I. Cryo-EM structures of assembly intermediates have helped us elucidate the role of NDUFAF1 in building the proton pumping module and of NDUFAF2 in attaching the NADH oxidation module. Our second goal is to understand complex I assembly and the function of the assembly factors in each step. Our work on the structure, function and assembly of complex I has offered clues on the pathogenesis of NDUFS4-linked Leigh syndrome. Our third goal is to take advantage of our yeast genetic model system to unravel how complex I dysfunction causes human diseases.
P15 Structure and mechanism of membrane-bound hydrogenases
PI: Bonnie J. Murphy
Group IV membrane-bound hydrogenases (MBHs) are members of the Complex I superfamily, catalyzing H2 production coupled with ion translocation across a membrane by an unknown mechanism. Using biochemical techniques and anaerobic, redox-controlled single-particle cryo-EM, we will study both the structure and mechanism of two MBHs: the formate-hydrogen lyase (FHL) complex from E. coli, and the energy-converting hydrogenase (Ech) from methanogenic archaea. By solving structures of the complexes under different gas and redox conditions, we will gain insights into the conformational changes that accompany catalytic turnover, and therefore the mechanism of coupling between substrate turnover and ion translocation.
P16 Dissecting the role of SND pathway components in ER membrane protein insertion
PI: Melanie McDowell
The signal recognition particle independent (SND) pathway targets and inserts a subset of integral membrane proteins into the endoplasmic reticulum membrane, requiring three component proteins: Snd1, Snd2 and Snd3. Using combined structural, biophysical and in vitro reconstitution approaches, we propose to define the precise molecular roles of these proteins and understand how they interact within the SND pathway.
P17 Understanding nuclear pore complexity
Nuclear pore complexes (NPC) bridge the nuclear membranes and mediate nucleocytoplasmic exchange. The central channel of the NPC is lined with intrinsically disordered, FG-rich nucleoporins (Nups) that interact with cargos. How exactly those FG-Nups distribute within the central channel and how they may adapt to conformational changes of the NPC remains unknown. In project P17, we propose to develop residue specific labeling methods for correlative light and electron microscopy to address this challenge. This novel method will allow to jointly measure the conformations of intrinsically disordered FG-domains and structured Nups.
P18 Protein assemblies and machineries in antigen processing and ER quality control
PI: Robert Tampé
Project P18 investigates cellular machineries in the adaptive immune system and and quality control in organellar membranes during viral infection and malignant transformation. The mechanistic principles of assembly and disassembly, conformational coupling, supramolecular organization, and modulation of the peptide-loading complex (PLC) by viral and cellular factors will be studied by integrative biochemical and biophysical approaches.