title: Direct Identification of Protein-Protein Interactions by Single-Molecule Force Spectroscopy.
abstract: 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.
authors.affiliation: Instituto Cajal, Consejo Superior de Investigaciones Científicas, Avda. Doctor Arce 37, E-, 28002, Madrid, Spain. avera@cajal.csic.es.|Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA-Nanociencia), E-, 28049, Cantoblanco, Madrid, Spain. avera@cajal.csic.es.
authors.forename: Andrés M
authors.initials: AM
authors.lastname: Vera
authors.affiliation: Instituto Cajal, Consejo Superior de Investigaciones Científicas, Avda. Doctor Arce 37, E-, 28002, Madrid, Spain. mcarrion@cajal.csic.es.|Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA-Nanociencia), E-, 28049, Cantoblanco, Madrid, Spain. mcarrion@cajal.csic.es.
authors.forename: Mariano
authors.initials: M
authors.lastname: Carrión-Vázquez
mesh_terms: D018797:Cell Cycle Proteins / Q000737:chemistry*; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D011485:Protein Binding; D000097722:Cohesins
chemical_list: D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D000097722:Cohesins
keywords: atomic force microscopy; biophysical methods; polyproteins; protein-protein interactions; single-molecule force spectroscopy