title: Force dependency of biochemical reactions measured by single-molecule force-clamp spectroscopy.
abstract: Here we describe a protocol for using force-clamp spectroscopy to precisely quantify the effect of force on biochemical reactions. A calibrated force is used to control the exposure of reactive sites in a single polyprotein substrate composed of repeated domains. The use of polyproteins allows the identification of successful single-molecule recordings from unambiguous mechanical unfolding fingerprints. Biochemical reactions are then measured directly by detecting the length changes of the substrate held at a constant force. We present the layout of a force-clamp spectrometer along with protocols to design and conduct experiments. These experiments measure reaction kinetics as a function of applied force. We show sample data of the force dependency of two different reactions, protein unfolding and disulfide reduction. These data, which can be acquired in just a few days, reveal mechanistic details of the reactions that currently cannot be resolved by any other technique.
authors.affiliation: Department of Biological Sciences, Columbia University, New York, USA. ivp2105@columbia.edu
authors.forename: Ionel
authors.initials: I
authors.lastname: Popa
authors.forename: Pallav
authors.initials: P
authors.lastname: Kosuri
authors.forename: Jorge
authors.initials: J
authors.lastname: Alegre-Cebollada
authors.forename: Sergi
authors.initials: S
authors.lastname: Garcia-Manyes
authors.forename: Julio M
authors.initials: JM
authors.lastname: Fernandez
mesh_terms: D002138:Calibration; D004220:Disulfides / Q000737:chemistry; D004867:Equipment Design; D007700:Kinetics; D018625:Microscopy, Atomic Force / Q000295:instrumentation / Q000379:methods*; D058767:Protein Unfolding
chemical_list: D004220:Disulfides