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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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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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)
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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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DOI: https://doi.org/10.1038/nmeth851
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