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Nat Commun. 2013;4:2660. doi: 10.1038/ncomms3660.

Visualization and targeted disruption of protein interactions in living cells.

Author information

1
1] Department of Biology, Technische Universität Darmstadt, 64287 Darmstadt, Germany [2].

Abstract

Protein-protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein-protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53-HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein-protein interactions in practically any cell type and species.

PMID:
24154492
PMCID:
PMC3826628
DOI:
10.1038/ncomms3660
[Indexed for MEDLINE]
Free PMC Article

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