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Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR)

Nature Methods volume 3, pages 91–93 (2006)Cite this article

Abstract

We describe a high-throughput in-cell nuclear magnetic resonance (NMR)-based method for mapping the structural changes that accompany protein-protein interactions (STINT-NMR). The method entails sequentially expressing two (or more) proteins within a single bacterial cell in a time-controlled manner and monitoring the protein interactions using in-cell NMR spectroscopy. The resulting spectra provide a complete titration of the interaction and define structural details of the interacting surfaces at atomic resolution.

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Figure 1: Overview of STINT-NMR methodology.
Figure 2: NMR spectra and interaction maps of ubiquitin-ligand complexes.

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Acknowledgements

This work was supported by grants to A.S. (American Diabetes Association Career Development Award 1-06-CD-23) and to D.C. (National Institutes of Health 2R01GM047021-13A2).

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Authors and Affiliations

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

    David S Burz & Alexander Shekhtman

  2. New York Structural Biology Center, 89 Convent Avenue, Park Building at 133rd St., New York, 10027, 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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  4. Alexander Shekhtman
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Correspondence to Alexander Shekhtman.

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Supplementary information

Supplementary Fig. 1

SDS-PAGE of ubiquitin and STAM2 sequential expression. (PDF 207 kb)

Supplementary Fig. 2

Overlay of 1H{15N}HSQC-spectra of E. coli cells after 3 hour over-expression of [U-, 15N] AUIM and 0 h (red), 2h (yellow), and 3 h (blue) over-expression of ubiquitin. (PDF 136 kb)

Supplementary Fig. 3

Overlay of 1H{15N}HSQC-spectra of E. coli cells after 3 hour over-expression of [U-, 15N] ubiquitin (red) and purified [U-, 15N] ubiquitin (black) in 10 mM phosphate buffer [pH 6.8]. (PDF 146 kb)

Supplementary Fig. 4

Overlay of 1H{15N}HSQC-spectra of E. coli cells after 3 hour over-expression of [U-, 15N] ubiquitin and 0 h (red), 2h (blue), and 3 h (yellow) over-expression of mutant AUIM (SA16). (PDF 117 kb)

Supplementary Fig. 5

Differential broadening of selected peaks from the 1H{15N}HSQC-spectrum of E. coli cells after 3 hour overexpression of [U-, 15N] ubiquitin and 0 h (red), 2 h (blue), and 3 h (green) overexpression of STAM2. (PDF 195 kb)

Supplementary Fig. 6

1H{15N}HSQC-spectrum of the supernatant of the NMR sample after E. coli cells over-expressing [U-15N] ubiquitin were removed by centrifugation. (PDF 104 kb)

Supplementary Fig. 7

1H{15N}HSQC-spectrum of the E. coli cells grown on [U-15N] M9 medium without protein over-expression. (PDF 89 kb)

Supplementary Methods (DOC 41 kb)

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Burz, D., Dutta, K., Cowburn, D. et al. Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR). Nat Methods 3, 91–93 (2006). https://doi.org/10.1038/nmeth851

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