Research Areas

 

In-situ Electron Spin Resonance (ESR) spectroscopy

 

Membrane transport mechanisms

 

Outer membrane Biogenesis:

 

i) β-Barrel Assembly Machinery (BAM)

 

ii) Lipopolysaccharide Transport (Lpt) system

 

 

In-situ Electron Spin Resonance (ESR) spectroscopy - watching proteins in cells and native membranes

Membrane proteins often transverse through a broad energy landscape and undergo large conformational changes during function. The dream for a structural biologist is to observe them in the cellular environment. However, reconstruction of a membrane protein structure in-situ with sufficient resolution has not been possible yet. Over the past few years, we developed an in-situ pulsed ESR spectroscopy approach (DEER/PELDOR) for observing the structure and conformational changes of outer membrane protein complexes in the native membrane and intact E. coli. One of the major focuses our laboratory is to further improve this methodology as well as to develop a similar approach for the a-helical inner membrane proteins. In this respect, we employ new spin labels, labeling strategies, sample preparation protocols, and advanced pulse sequences. 

 

 

 

 

 

 

 

 

 

 

 

 

 

Related Publications:

  1. Joseph B*, Jaumann EA, Sikora A, Barth K, Prisner TF, Cafiso DS (2019In-situ observation of conformational dynamics and protein-ligand/substrate interaction in outer membrane proteins with DEER/PELDOR spectroscopy. Nat. Protoc., In press (*corresponding author)

  2. Joseph B*, Tormyshev VM, Rogozhnikova OYu, Akhmetzyanov D, Bagryanskaya EG, Prisner TF* (2016) Selective High-Resolution Detection of Membrane Protein-Ligand Interaction in Native Membranes using Trityl-Nitroxide PELDOR. Angew. Chem. Int. Ed., 55, 11538-11542. (*corresponding authors)

  3. Joseph B*, Sikora A, Cafiso DS* (2016) Ligand-induced conformational changes in a membrane transporter in E. coli cells observed with DEER/PELDOR. J. Am. Chem. Soc., 138, 1844-1847. (highlighted in JACS Spotlights; *corresponding authors)

  4. Sikora A, Joseph B, Matson M, Staley JR, Cafiso DS (2016) Allosteric Signaling Is Bidirectional in an Outer-Membrane Transport Protein. Biophys J., 111, 1908-1918.

  5. Joseph B, Sikora A, Bordignon E, Jeschke G, Cafiso DS, Prisner TF (2015) Distance Measurement on an Endogenous Membrane Transporter in E. coli Cells and Native Membranes Using EPR Spectroscopy. Angew. Chem. Int. Ed.54, 6196-6199.

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Membrane transport mechanisms

The function of cells and organelle depend on the transport of diverse molecules across the biological membrane. When the molecules move along a concentration gradient, the chemical potential energy itself could drive the transport. However, membrane transporters often carry substrates against a concentration gradient. In order to overcome such an energy barrier, they couple an external energy source with substrate translocation. Depending on the transporter, the energy may be provided by ATP, electrochemical gradient, or light. In our group, we study ATP Binding Cassette (ABC) transporters (in collaboration with Tampé lab) and a proton-coupled secondary transporter (SLC26Dg, in collaboration with Geertsma lab). Specifically, we address how the energy of ATP hydrolysis or proton gradient is coupled to conformational changes and substrate translocation.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                                                                                                                                                                                                                                  

Related Publications:

  1. Chang YN, Jaumann EA, Reichel K, Hartmann J, Oliver D, Hummer G*, Joseph B*, Geertsma ER* (2019) Structural basis for functional interaction in dimers of SLC26 transporters. Nat. Commun., in press (*corresponding authors)

  2. Barth K, Hank S, Spindler PE, Prisner TF, Tampé R, Joseph B* (2018) Conformational Coupling and trans-Inhibition in the Human Antigen Transporter Ortholog TmrAB Resolved with Dipolar EPR Spectroscopy.            J. Am. Chem. Soc., 140, 4527–4533. (*corresponding author)

  3. Bock C, Löhr F, Tumulka F, Reichel K, Würz J, Hummer G, Schäfer L, Tampé R, Joseph B, Bernhard F, Dötsch V, Abele R. Structural and functional insights into the interaction and targeting hub TMD0 of the polypeptide transporter TAPL (2018). Sci. Rep., 8(1):15662.

  4. Nöll A, Thomas C, Herbring V, Zollmann T, Barth K, Mehdipour AR, Tomasiak TM, Brüchert S, Joseph B, Abele R, Oliéric V, Wang M, Diederichs K, Hummer G, Stroud RM, Pos KM, and Tampé R (2017) Crystal structure and mechanistic basis of a functional homolog of the antigen transporter TAP. Proc. Natl. Acad. Sci. USA., 114,
    E438-447.

  5. Joseph B, Korkhov VM, Yulikov M, Jeschke G, Bordignon E (2014) Conformational cycle of the vitamin B12 ABC importer in liposomes detected by double electron-electron resonance (DEER). J. Biol. Chem., 289, 3176-3185.

  6. Doll A, Bordignon E, Joseph B, Tschaggelar R, Jeschke G (2012) Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures. J. Magn. Reson., 222, 34-43.

  7. Joseph B, Jeschke G, Goetz BA, Locher KP, Bordignon E (2011) Transmembrane gate movements in type II ATP-binding cassette (ABC) importer BtuCD-F during nucleotide cycle. J. Biol. Chem., 286, 41008-41017.

                                                                                                                                                         

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Outer membrane biogenesis in Gram-negative bacteria

Gram-negative bacteria have become increasingly resistant to available antibiotics resulting in more illness, healthcare costs, and deaths. Their cell envelope consists of the inner membrane (IM) surrounding the cytoplasm and an outer membrane (OM) that protects the cells from harsh conditions. The OM is an asymmetric bilayer made up of phospholipids (PL) and lipopolysaccharides (LPS). Also, the OM harbors numerous β- barrel proteins (outer membrane proteins, OMPs). Both LPS and OMPs are synthesized in the cytoplasm and subsequently transported across the periplasm into the OM. In E. coli,  seven essential proteins LptABCDEFG spanning the entire cell envelope form the LPS transport system. Similarly, The β-Barrel Assembly Machinery (BAM), which consists of the BamABCDE subunits mediates folding and insertion of OMP precursors from periplasm into the OM.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The BAM complex - β-barrel folding and insertion mechanism

In the two available structures for the BAM full-complex, the central β-barrel BamA exists either in an inward-open or lateral open conformation. It is considered that those conformational changes might be coupled to OMP folding and insertion through an unknown mechanism. As the asymmetric outer membrane is an integral part of the BAM complex, mechanistic investigations must be performed in whole cells or native membranes. Using the in-situ ESR approach, which we demonstrated over the past years, we are elucidating how the conformational changes in BAM are coupled to protein folding and insertion.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The Lpt System - lipopolysaccharide (LPS) transport mechanism

The Lpt system transports LPS molecules from the inner membrane to the outer membrane through the tans-periplasmic bridge consisting of LptC, LptA, and the N-terminal domain of LptD. The ABC exporter LptB2FG located in the inner membrane is suggested to extract LPS molecules and push it through the periplasm at the expense of ATP hydrolysis. Structures of the individual subunits have been resolved. We investigate how the subunits interact with each other to form the supramolecular trans-envelope complex and also how ATP hydrolysis at the inner membrane powers LPS transport through the periplasmic bridge into the outer membrane.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  

                                                                                                                                                                 

                                                                                                                                                                                                                                       To top