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Biophys J. 2012 Oct 3;103(7):1490-9. doi: 10.1016/j.bpj.2012.06.051. Epub 2012 Oct 2.

Limitations of constant-force-feedback experiments.

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1
Biophysics Graduate Group, University of California, Berkeley, CA, USA.

Abstract

Single-molecule force spectroscopy has provided important insights into the properties and mechanisms of biological molecules and systems. A common experiment is to measure the force dependence of conformational changes at equilibrium. Here, we demonstrate that the commonly used technique of force feedback has severe limitations when used to evaluate rapid macromolecular conformational transitions. By comparing the force-dependent dynamics of three major classes of macromolecules (DNA, RNA, and protein) using both a constant-force-feedback and a constant-trap-position technique, we demonstrate a problem in force-feedback experiments. The finite response time of the instrument's force feedback can modify the behavior of the molecule, leading to errors in the reported parameters, such as the rate constants and the distance to the transition state, for the conformational transitions. We elucidate the causes of this problem and provide a simple test to identify and evaluate the magnitude of the effect. We recommend avoiding the use of constant force feedback as a method to study rapid conformational changes in macromolecules.

PMID:
23062341
PMCID:
PMC3471466
DOI:
10.1016/j.bpj.2012.06.051
[Indexed for MEDLINE]
Free PMC Article
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