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

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

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Figure 17.11.1 Flow chart to generate a matrix of samples for STINT‐NMR analysis.Reprinted from Burz et al. ()

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Figure 17.11.2 SDS‐PAGE of ubiquitin and STAM2 sequential expression. The BL21(DE3) cells were analyzed immediately after overexpression; lane 1 is uninduced cells; lanes 2 to 5 are a timecourse of ubiquitin overexpression induced using L ‐arabinose for 0.5, 1, 2, and 3 hr, respectively; lanes 6 to 9 are sequential overexpression of STAM2 induced using IPTG for 0.5, 1, 2, and 3 hr, respectively. Note that the ubiquitin level remains essentially constant as STAM2 expression increases. Reprinted from Burz et al. (). Abbreviations: ubq, ubiquitin

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Figure 17.11.3 NMR spectra of the target protein, ubiquitin, complexed with interactor peptide, AUIM, and with interactor protein, STAM2. (A ) Overlay of 1 H{15 N}HSQC spectra of E. coli cells after 3 hr of overexpressing [ U ‐, 15 N] ubiquitin and 0 hr (black), 2 hr (red), and 3 hr (blue) of overexpressing AUIM. Individual peaks exhibiting large chemical shifts are labeled with corresponding assignments. The progression of colors in the 1 H{15 N}HSQC overlaid spectra was chosen for ease of viewing. Inset: Close‐up of the chemical shift changes of Gly47 during titration. (B ) 1 H{15 N}HSQC spectra of E. coli cells after 3 hr of overexpressing [ U ‐, 15 N] ubiquitin and 3 hr of overexpressing STAM2. Resonance peaks exhibiting extreme broadening are indicated by crosses. Insets: One‐dimensional traces of selected peaks exhibiting differential broadening after 3 hr of overexpressing [ U‐ , 15 N] ubiquitin and 0 hr (black), 2 hr (red), and 3 hr (green) of overexpressing STAM2. Reprinted from Burz et al. ()

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Figure 17.11.4 Interaction surface maps of ubiquitin‐ligand complexes. Interaction surface of ubiquitin mapped onto the three‐dimensional structure of ubiquitin (PDB code 1D3Z). Individual residues exhibiting either a chemical shift change >0.05 ppm or differential broadening are indicated in red. All perturbed residues lie on the ubiquitin surface and, therefore, reflect changes in the interaction surface of the molecule rather than changes in tertiary or quaternary structure. (A ) Y371/4F‐STAM2‐Ubq interaction; (B ) phosphorylated Y371/4F‐STAM2‐Ubq interaction (YP‐Y371/4F‐STAM2). Ubiquitin ligands are indicated in each panel. This interaction map was used to prove that Y371/4F mutation leads to a loss of the dependence of STAM2‐ubiquitin interaction on the phosphorylation state of STAM2. Reprinted from Burz and Shekhtman ()

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Figure 17.11.5 Matrix method of screening chemical libraries. A library containing 289 dipeptide compounds is screened by examining individual mixtures located in the first row and first column of a matrix plate. Mixtures that result in similar changes in the in‐cell NMR spectrum, so called hits, located at the intersection of rows (red) and columns (blue), are used in the second round of screening to validate the hit. Reprinted with permission from Xie et al ()

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