The STINT‐NMR Method for Studying In‐cell Protein‐Protein Interactions

David S. Burz1, Alexander Shekhtman1

1 State University of New York at Albany, Albany, New York
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 17.11
DOI:  10.1002/0471140864.ps1711s61
Online Posting Date:  August, 2010
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Abstract

This unit describes critical components and considerations required to study protein‐protein structural interactions inside a living cell by using NMR spectroscopy (STINT‐NMR). STINT‐NMR 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 spectra provide a complete titration of the interaction and define structural details of the interacting surfaces at the level of single amino acid residues. The advantages and limitations of STINT‐NMR are discussed, along with the differences between studying macromolecular interactions in vitro and in vivo (in‐cell). Also described are considerations in the design of STINT‐NMR experiments, focusing on selecting appropriate overexpression plasmid vectors, sample requirements and instrumentation, and the analysis of STINT‐NMR data, with specific examples drawn from published works. Applications of STINT‐NMR, including an in‐cell methodology to post‐translationally modify interactor proteins and an in‐cell NMR assay for screening small molecule interactor libraries (SMILI‐NMR) are presented. Curr. Protoc. Protein Sci. 61:17.11.1‐17.11.15. © 2010 by John Wiley & Sons, Inc.

Keywords: in‐cell biochemistry; in‐cell NMR spectroscopy; protein‐protein interactions; drug screening; proteomics

     
 
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Table of Contents

  • Introduction
  • Advantages and Limitations
  • Experimental Design
  • Sample Requirements and Handling
  • Data and Analysis
  • Applications
  • SMILI‐NMR
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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Literature Cited

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