Force dependency of biochemical reactions measured by single-molecule force-clamp spectroscopy

• Published in: Nature protocols Vol. 8; no. 7; pp. 1261 - 1276
• Main Authors: Popa, Ionel, Kosuri, Pallav, Alegre-Cebollada, Jorge, Garcia-Manyes, Sergi, Fernandez, Julio M
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.07.2013; Nature Publishing Group
• Subjects: 631/1647/527; 631/45/612; 631/57/2265; Analysis; Analytical Chemistry; Atomic spectroscopy; Biochemistry; Biological Techniques; Calibration; Chemical bonds; Chemical kinetics; Chemical reactions; Computational Biology/Bioinformatics; Data acquisition; Disulfides - chemistry; Equipment Design; Experiments; Intermolecular forces; Kinetics; Life Sciences; Microarrays; Microscopy, Atomic Force - instrumentation; Microscopy, Atomic Force - methods; Organic Chemistry; Polyproteins; Protein denaturation; Protein folding; Protein Unfolding; Proteins; Protocol; Reaction kinetics; Spectroscopy; Spectrum analysis; Substrates; United States; United Kingdom > UK

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 i...