Abstract
Molecular machines are the workhorses of the cell that efficiently convert chemical energy into mechanical motion through conformational changes. They can be considered powerful machines, exerting forces and torque on the molecular level of several piconewtons and piconewton-nanometer, respectively. For studying translocation and conformational changes of these machines, fluorescence methods, like FRET, as well as “mechanical” methods, like optical and magnetic tweezers, have proven well suited over the past decades. One of the current challenges in the field of molecular machines is gaining maximal information from single-molecule experiments by simultaneously measuring translocation, conformational changes, and forces exerted by these machines. In this chapter, we describe the combination of magnetic tweezers with single-molecule FRET for orthogonal simultaneous readout to maximize the information gained in single-molecule experiments.
Keywords
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- CCD:
-
Charge-coupled device
- DM:
-
Dichroic mirror
- DNA:
-
Deoxyribonucleic acid
- dNTP:
-
Deoxyribonucleoside triphosphate
- dTTP:
-
Deoxythymidine triphosphate
- dUTP:
-
Deoxyuridine triphosphate
- EMCCD:
-
Electron multiplying charge-coupled device
- FRET:
-
Förster resonance energy transfer
- GPU:
-
Graphics processing unit
- HJ:
-
Holliday junction
- LUT:
-
Lookup table
- PCR:
-
Polymerase chain reaction
- RCLED:
-
Resonant-cavity light-emitting diode
- RNA:
-
Ribonucleic acid
- ROI:
-
Region of interest
- TIRF:
-
Total internal reflection fluorescence
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Acknowledgements
We gratefully acknowledge numerous discussions with the group of Ralf Seidel, in particular Ralf Seidel, Alexander Huhle, and Friedrich Schwarz. Furthermore, discussions and software support by members of the Schlierf lab are highly appreciated. This work was supported by grants from BMBF 03Z2EN11 (to M.S.).
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Swoboda, M., Grieb, M.S., Hahn, S., Schlierf, M. (2014). Measuring Two at the Same Time: Combining Magnetic Tweezers with Single-Molecule FRET. In: Toseland, C., Fili, N. (eds) Fluorescent Methods for Molecular Motors. Experientia Supplementum, vol 105. Springer, Basel. https://doi.org/10.1007/978-3-0348-0856-9_12
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