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In Situ Observation of Conformational Dynamics and Protein Ligand–Substrate Interactions in Outer-Membrane Proteins With DEER/PELDOR Spectroscopy
Nature Protocols
  • Benesh Joseph, University of Frankfurt - Germany
  • Eva A. Jaumann, University of Frankfurt - Germany
  • Arthur Sikora, University of Virginia
  • Katja Barth, University of Frankfurt - Germany
  • Thomas F. Prisner, University of Frankfurt - Germany
  • David Cafiso, University of Virginia
Document Type
Article
Publication Date
8-1-2019
Abstract

Observation of structure and conformational dynamics of membrane proteins at high resolution in their native environments is challenging because of the lack of suitable techniques. We have developed an approach for high-precision distance measurements in the nanometer range for outer-membrane proteins (OMPs) in intact Escherichia coli and native membranes. OMPs in Gram-negative bacteria rarely have reactive cysteines. This enables in situ labeling of engineered cysteines with a methanethiosulfonate spin label (MTSL) with minimal background signals. Following overexpression of the target protein, spin labeling is performed with E. coli or isolated outer membranes (OMs) under selective conditions. The interspin distances are measured in situ, using pulsed electron–electron double resonance (PELDOR or DEER) spectroscopy. The residual background signals, which are problematic for in situ structural biology, contribute specifically to the intermolecular part of the signal and can be selectively removed to extract the desired interspin distance distribution. The initial cloning stage can take 5–7 d, and the subsequent protein expression, OM isolation, spin labeling, PELDOR experiment, and data analysis typically take 4–5 d. The described protocol provides a general strategy for observing protein ligand–substrate interactions, oligomerization, and conformational dynamics of OMPs in their native OM and intact E. coli.

Comments

©2019 Springer Nature Limited

Additional Comments
Marie-Curie COFUND Postdoctoral program #: PCOFUND-GA-2011-291776, GO-IN; Deutsche Forschungsgemeinschaft grant #: SFB807; NIH grant #: GM035215
ORCID ID
0000-0001-6295-9928
DOI
10.1038/s41596-019-0182-2
Citation Information
Benesh Joseph, Eva A. Jaumann, Arthur Sikora, Katja Barth, et al.. "In Situ Observation of Conformational Dynamics and Protein Ligand–Substrate Interactions in Outer-Membrane Proteins With DEER/PELDOR Spectroscopy" Nature Protocols Vol. 14 (2019) p. 2344 - 2369 ISSN: 1754-2189
Available at: http://works.bepress.com/arthur-sikora/22/