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PLoS One. 2014 Dec 29;9(12):e115049. doi: 10.1371/journal.pone.0115049. eCollection 2014.

Parallel force assay for protein-protein interactions.

Author information

1
Lehrstuhl für Angewandte Physik and Center for Nanoscience (CeNS), Ludwig-Maximilians-; Munich Center for Integrated Protein Science (CIPSM), Butenandtstr. 5-13, 81377 Munich, Germany.
2
Lehrstuhl für Angewandte Physik and Center for Nanoscience (CeNS), Ludwig-Maximilians-
3
Lehrstuhl für Angewandte Physik and Center for Nanoscience (CeNS), Ludwig-Maximilians-; Department of Biology II and Center for Nanoscience (CeNS), Ludwig-Maximilians-Universität, Großhadernerstr. 2, 82152 Planegg-Martinsried, Germany.
4
Department of Biology II and Center for Nanoscience (CeNS), Ludwig-Maximilians-Universität, Großhadernerstr. 2, 82152 Planegg-Martinsried, Germany; Munich Center for Integrated Protein Science (CIPSM), Butenandtstr. 5-13, 81377 Munich, Germany.

Abstract

Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.

PMID:
25546146
PMCID:
PMC4278885
DOI:
10.1371/journal.pone.0115049
[Indexed for MEDLINE]
Free PMC Article

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