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 unfol

SUBMITTER: Popa I 

PROVIDER: S-EPMC4676941 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature