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Small. 2015 Mar 18;11(11):1273-84. doi: 10.1002/smll.201402686. Epub 2014 Nov 14.

Probing DNA helicase kinetics with temperature-controlled magnetic tweezers.

Author information

1
Centro Nacional de Biotecnología, CSIC, Darwin 3, Campus de Cantoblanco, 28049, Madrid, Spain.

Abstract

Motor protein functions like adenosine triphosphate (ATP) hydrolysis or translocation along molecular substrates take place at nanometric scales and consequently depend on the amount of available thermal energy. The associated rates can hence be investigated by actively varying the temperature conditions. In this article, a thermally controlled magnetic tweezers (MT) system for single-molecule experiments at up to 40 °C is presented. Its compact thermostat module yields a precision of 0.1 °C and can in principle be tailored to any other surface-coupled microscopy technique, such as tethered particle motion (TPM), nanopore-based sensing of biomolecules, or super-resolution fluorescence imaging. The instrument is used to examine the temperature dependence of translocation along double-stranded (ds)DNA by individual copies of the protein complex AddAB, a helicase-nuclease motor involved in dsDNA break repair. Despite moderately lower mean velocities measured at sub-saturating ATP concentrations, almost identical estimates of the enzymatic reaction barrier (around 21-24 k(B)T) are obtained by comparing results from MT and stopped-flow bulk assays. Single-molecule rates approach ensemble values at optimized chemical energy conditions near the motor, which can withstand opposing loads of up to 14 piconewtons (pN). Having proven its reliability, the temperature-controlled MT described herein will eventually represent a routinely applied method within the toolbox for nano-biotechnology.

KEYWORDS:

AddAB helicase-nuclease; activation energy; magnetic tweezers; single-molecule studies; temperature control

PMID:
25400244
PMCID:
PMC4473356
DOI:
10.1002/smll.201402686
[Indexed for MEDLINE]
Free PMC Article

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