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

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...

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Bibliographic Details
Published inNature 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
LanguageEnglish
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
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Summary: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.
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These authors contributed equally to the work.
ISSN:1754-2189
1750-2799
1750-2799
DOI:10.1038/nprot.2013.056