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Measuring Two at the Same Time: Combining Magnetic Tweezers with Single-Molecule FRET

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Fluorescent Methods for Molecular Motors

Part of the book series: Experientia Supplementum ((EXS,volume 105))

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.

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Abbreviations

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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Authors and Affiliations

  1. BCUBE - Center for Molecular Bioengineering, Technische Universität Dresden, Arnoldstraße 18, 01307, Dresden, Germany

    Marko Swoboda, Maj Svea Grieb, Steffen Hahn & Michael Schlierf

Authors
  1. Marko Swoboda
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  2. Maj Svea Grieb
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  3. Steffen Hahn
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  4. Michael Schlierf
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Corresponding author

Correspondence to Michael Schlierf .

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Editors and Affiliations

  1. Chromosome Organisation and Dynamics, Max-Planck Institute of Biochemistry, Martinsried, Germany

    Christopher P. Toseland

  2. Department of Cellular Physiology, Ludwig-Maximilians-Universität München, Munich, Germany

    Natalia Fili

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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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