Journal
SMALL
Volume 11, Issue 11, Pages 1273-1284Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/smll.201402686
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Funding
- European Research Council [206117]
- Spanish Ministry of Science and Innovation (MICINN) [FIS2011-24638]
- Wellcome Trust [077368, 100401]
- Spanish Ministry of Education
- Juan de la Cierva Program [MICINN] [JCI-2011-10277]
- BBSRC
- European Research Council (ERC) [206117] Funding Source: European Research Council (ERC)
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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 degrees C is presented. Its compact thermostat module yields a precision of 0.1 degrees 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.
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