Premium
Direct Identification of Protein–Protein Interactions by Single‐Molecule Force Spectroscopy
Author(s) -
Vera Andrés M.,
CarriónVázquez Mariano
Publication year - 2016
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201605284
Subject(s) - force spectroscopy , intermolecular force , intramolecular force , molecule , chemistry , chemical physics , atomic force microscopy , protein–protein interaction , covalent bond , dissociation (chemistry) , spectroscopy , crystallography , biophysics , nanotechnology , materials science , stereochemistry , physics , biochemistry , biology , organic chemistry , quantum mechanics
Single‐molecule force spectroscopy based on atomic force microscopy (AFM‐SMFS) has allowed the measurement of the intermolecular forces involved in protein‐protein interactions at the molecular level. While intramolecular interactions are routinely identified directly by the use of polyprotein fingerprinting, there is a lack of a general method to directly identify single‐molecule intermolecular unbinding events. Here, we have developed an internally controlled strategy to measure protein–protein interactions by AFM‐SMFS that allows the direct identification of dissociation force peaks while ensuring single‐molecule conditions. Single‐molecule identification is assured by polyprotein fingerprinting while the intermolecular interaction is reported by a characteristic increase in contour length released after bond rupture. The latter is due to the exposure to force of a third protein that covalently connects the interacting pair. We demonstrate this strategy with a cohesin–dockerin interaction.