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

David S. Burz, Kaushik Dutta, David Cowburn, Alexander Shekhtman

Research output: Contribution to journalArticle

62 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)146-152
Number of pages7
JournalNature Protocols
Volume1
Issue number1
DOIs
StatePublished - Jun 1 2006

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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