期刊
METHODS
卷 108, 期 -, 页码 24-39出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2016.04.028
关键词
Kinetics; Thermodynamics; Mechanism; ATP; NTP; Hydrolysis; Translocation; Unwinding; Nucleic acid; DNA; RNA; Helicase; Translocase
资金
- Marie Sklodowska-Curie Reintegration Fellowship [H2020-MSCA-IF-2014-657076]
- Momentum Grant of the Hungarian Academy of Sciences [LP2011-006/2011, NKFIH K-117062]
Helicases and translocases are nucleic acid (NA)-based molecular motors that use the free energy liberated during the nucleoside triphosphate (NTP, usually ATP) hydrolysis cycle for unidirectional translocation along their NA (DNA, RNA or heteroduplex) substrates. Determination of the kinetic and thermodynamic parameters of their mechanoenzymatic cycle serves as a basis for the exploration of their physiological behavior and various cellular functions. Here we describe how recent adaptations of fluorescence -based solution kinetic methods can be used to determine practically all important mechanistic parameters of NA -based motor proteins. We outline practically useful analysis procedures for equilibrium, steady-state and transient kinetic data. This analysis can be used to quantitatively characterize the enzymatic steps of the NTP hydrolytic cycle, the binding site size, stoichiometry and energetics of protein-NA interactions, the rate and processivity of translocation along and unwinding of NA strands, and the mechanochemical coupling between these processes. The described methods yield insights into the functional role of the enzymes, and also greatly aid the design and interpretation of single-molecule experiments as well as the engineering of enzymatic properties for biotechnological applications. (C) 2016 Elsevier Inc. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据