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In-cell NMR for protein-protein interactions (STINT-NMR)

Nature Protocols volume 1, pages 146–152 (2006)Cite this article

Abstract

We describe an in-cell NMR-based method for mapping the structural interactions (STINT-NMR) that underlie protein-protein complex formation. This method entails sequentially expressing two (or more) proteins within a single bacterial cell in a time-controlled manner and monitoring their interactions using in-cell NMR spectroscopy. The resulting NMR data provide a complete titration of the interaction and define structural details of the interacting surfaces at atomic resolution. Unlike the case where interacting proteins are simultaneously overexpressed in the labeled medium, in STINT-NMR the spectral complexity is minimized because only the target protein is labeled with NMR-active nuclei, which leaves the interactor protein(s) cryptic. This method can be combined with genetic and molecular screens to provide a structural foundation for proteomic studies. The protocol takes 4 d from the initial transformation of the bacterial cells to the acquisition of the NMR spectra.

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Figure 1: Flowchart to generate samples for STINT-NMR analysis.
Figure 2: NMR spectra and interaction maps of the target protein, ubiquitin, complexed with interactor peptide AUIM and with interactor protein STAM2.

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Acknowledgements

This work was supported by grants to A.S. (American Diabetes Association Career Development Award) and to D.C. (National Institutes of Health).

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Authors and Affiliations

  1. Department of Chemistry, State University of New York at Albany, 1400 Washington Avenue, Albany, 12222, New York, USA

    David S Burz & Alexander Shekhtman

  2. New York Structural Biology Center, 89 Convent Avenue, Park Building at 133rd Street, New York, 10027-7566, New York, USA

    Kaushik Dutta & David Cowburn

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  1. David S Burz
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  2. Kaushik Dutta
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  3. David Cowburn
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  4. Alexander Shekhtman
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Correspondence to Alexander Shekhtman.

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Burz, D., Dutta, K., Cowburn, D. et al. In-cell NMR for protein-protein interactions (STINT-NMR). Nat Protoc 1, 146–152 (2006). https://doi.org/10.1038/nprot.2006.23

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