Probing DNA helicase kinetics with temperature-controlled magnetic tweezers.
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.
Centro Nacional de Biotecnología, CSIC, Darwin 3, Campus de Cantoblanco, 28049, Madrid, Spain.
Benjamin
B
Gollnick
Carolina
C
Carrasco
Francesca
F
Zuttion
Neville S
NS
Gilhooly
Mark S
MS
Dillingham
Fernando
F
Moreno-Herrero
D004247:DNA / Q000737:chemistry* / Q000648:ultrastructure; D004265:DNA Helicases / Q000737:chemistry* / Q000648:ultrastructure; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006361:Heating / Q000295:instrumentation; D008280:Magnetics / Q000295:instrumentation*; D008846:Micromanipulation / Q000295:instrumentation*; D008853:Microscopy / Q000295:instrumentation*; D015336:Molecular Probe Techniques / Q000295:instrumentation*; D011485:Protein Binding; D013314:Stress, Mechanical; D013696:Temperature
D004247:DNA; D004265:DNA Helicases
AddAB helicase-nuclease; activation energy; magnetic tweezers; single-molecule studies; temperature control