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Fluorescence to Study the ATPase Mechanism of Motor Proteins

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Book cover Fluorescent Methods for Molecular Motors

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

Abstract

This chapter provides an overview of different methodologies to dissect the ATPase mechanism of motor proteins. The use of ATP is fundamental to how these molecular engines work and how they can use the energy to perform various cellular roles. Rapid reaction and single-molecule techniques will be discussed to monitor reactions in real time through the application of fluorescence intensity, anisotropy and FRET. These approaches utilise fluorescent nucleotides and biosensors. While not every technique may be suitable for your motor protein, the different ways to determine the ATPase mechanism should allow a good evaluation of the kinetic parameters.

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Abbreviations

ATPγS:

Adenosine 5′-O-(3-thio)triphosphate

Deac:

Diethylaminocoumarin

dsDNA:

Double-stranded deoxyribonucleic acid

FRET:

Förster (or fluorescence) resonance energy transfer

k cat :

Catalytic turnover number

K d :

Equilibrium dissociation constant

K i :

Inhibition constant

K m :

Michaelis constant

Mant:

2′(3′)-O-(N-methylanthraniloyl)-

MDCC:

7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin

MDCC-PBP:

(A197C) Phosphate-binding protein adduct with MDCC

NADH:

Nicotinamide adenine dinucleotide

Pi :

Inorganic phosphate

ssDNA:

Single-stranded deoxyribonucleic acid

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Authors and Affiliations

  1. Chromosome Organisation and Dynamics, Max-Planck Institute of Biochemistry, Martinsried, 82152, Germany

    Christopher P. Toseland

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  1. Christopher P. Toseland
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Correspondence to Christopher P. Toseland .

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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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Toseland, C.P. (2014). Fluorescence to Study the ATPase Mechanism of Motor Proteins. 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_4

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