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

被引:95
作者
Popa, Ionel [1 ]
Kosuri, Pallav [1 ]
Alegre-Cebollada, Jorge [1 ]
Garcia-Manyes, Sergi [2 ,3 ]
Fernandez, Julio M. [1 ]
机构
[1] Columbia Univ, Dept Biol Sci, New York, NY 10027 USA
[2] Kings Coll London, Dept Phys, London WC2R 2LS, England
[3] Kings Coll London, Randall Div Cell & Mol Biophys, London WC2R 2LS, England
基金
瑞士国家科学基金会; 美国国家卫生研究院;
关键词
COVALENT IMMOBILIZATION; ENERGY LANDSCAPE; THIOREDOXIN CATALYSIS; MECHANICAL STABILITY; ELASTIC RESPONSE; PROTEIN; UBIQUITIN; KINETICS; BOND; AFM;
D O I
10.1038/nprot.2013.056
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:1261 / 1276
页数:16
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