title: Use of stopped-flow fluorescence and labeled nucleotides to analyze the ATP turnover cycle of kinesins.
abstract: The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. Kinesins display differences across the superfamily both in ATP turnover and in microtubule interaction. These differences tailor specific kinesins to various functions such as cargo transport, microtubule sliding, microtubule depolymerization and microtubule stabilization. To understand the mechanism of action of a kinesin it is important to understand how the chemical cycle of ATP turnover is coupled to the mechanical cycle of microtubule interaction. To dissect the ATP turnover cycle, one approach is to utilize fluorescently labeled nucleotides to visualize individual steps in the cycle. Determining the kinetics of each nucleotide transition in the ATP turnover cycle allows the rate-limiting step or steps for the complete cycle to be identified. For a kinesin, it is important to know the rate-limiting step, in the absence of microtubules, as this step is generally accelerated several thousand fold when the kinesin interacts with microtubules. The cycle in the absence of microtubules is then compared to that in the presence of microtubules to fully understand a kinesin's ATP turnover cycle. The kinetics of individual nucleotide transitions are generally too fast to observe by manually mixing reactants, particularly in the presence of microtubules. A rapid mixing device, such as a stopped-flow fluorimeter, which allows kinetics to be observed on timescales of as little as a few milliseconds, can be used to monitor such transitions. Here, we describe protocols in which rapid mixing of reagents by stopped-flow is used in conjunction with fluorescently labeled nucleotides to dissect the ATP turnover cycle of a kinesin.
authors.affiliation: School of Life Sciences, University of Nottingham.
authors.forename: Jennifer T
authors.initials: JT
authors.lastname: Patel
authors.affiliation: School of Life Sciences, University of Nottingham.
authors.forename: Hannah R
authors.initials: HR
authors.lastname: Belsham
authors.affiliation: School of Life Sciences, University of Nottingham.
authors.forename: Alexandra J
authors.initials: AJ
authors.lastname: Rathbone
authors.affiliation: School of Life Sciences, University of Nottingham; claire.friel@nottingham.ac.uk.
authors.forename: Claire T
authors.initials: CT
authors.lastname: Friel
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D016547:Kinesins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D009711:Nucleotides; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D016547:Kinesins