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 (DEER/PELDOR) approach 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 to develop a similar approach for the alpha-helical inner membrane proteins. In this respect, we employ new spin labels, labeling strategies, sample preparation protocols, and advanced pulse sequences.


Related Publications:

  1. A. Gopinath and B. Joseph* (2021) Conformational flexibility of the protein insertase BamA in the native asymmetric bilayer elucidated with ESR spectroscopy. Angew. Chem. Int. Ed., DOI: 10.1002/anie.202113448

  2. Anandi Kugele,  Sophie Ketter, Bjarne Silkenath,  Valentin Wittmann, Benesh Joseph,* Malte Drescher * (2021) In situ EPR Spectroscopy of a Bacterial Membrane Transporter using an Expanded Genetic Code. ChemComm, (accepted), DOI: 10.1039/d1cc04612h

  3. Ketter S, Gopinath A, Rogozhnikova O, Trukhin D, Tormyshev VM, Bhagryanskaya EG, Joseph B* (2021) In situ labeling and distance measurements of membrane proteins in E. coli using Finland and OX063 trityl labels. Chem. Eur. J., 27:2299-2304.

  4. Joseph B*, Jaumann EA, Sikora A, Barth K, Prisner TF, Cafiso DS (2019) In situ observation of conformational dynamics and protein-ligand/substrate interaction in outer membrane proteins with DEER/PELDOR spectroscopy. Nat. Protoc., 14:2344-2369. (*corresponding author)

  5. 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)

  6. 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)

  7. 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.

  8. 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.