Comparative single-molecule and ensemble myosin enzymology: Sulfoindocyanine ATP and ADP derivatives
Comparative single-molecule and ensemble myosin enzymology: Sulfoindocyanine ATP and ADP derivatives
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DOI：
10.1016/s0006-3495(00)76843-3
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发表时间：
2000-06-01
影响因子：
3.4
通讯作者：
Trentham, DR
中科院分区：
文献类型：
--
作者：
Oiwa, K;Eccleston, JF;Trentham, DR
比较了荧光核苷酸与肌球蛋白的单分子反应和宏观反应。单分子研究作为酶催化反应和配体-受体相互作用作为个体随机过程分析的范例。荧光核苷酸，称为Cy 3-EDA-ATP和Cy 5-EDA-ATP，通过将染料Cy3.29.OH和Cy5.29.OH偶联而衍生（化合物XI和XIV分别在Mujumdar等人（1993，Bioconjug. 4：105-111））与2 ′（3 ′）-O-[N-（2-氨乙基）氨基甲酰基]ATP（EDA-ATP）反应。ATP（ADP）类似物被分离成它们各自的2 '-和3'-O-异构体，在22 ℃时，它们的相互转化率为30[OH-] s（-1）（0.016 h（-1），pH7.1）。宏观研究表明，2 '（3'）-O-取代的核苷酸在与肌球蛋白、肌动球蛋白和肌纤维的相互作用中具有与ATP和ADP相似的性质，尽管ATP类似物不能像ATP那样松弛肌肉。Cy 3-核苷酸2 '-和3'-O-异构体在游离溶液中以及当它们与肌球蛋白相互作用时的荧光强度的显著差异是明显的。使用全内反射荧光显微镜的单分子研究表明，与肌球蛋白丝相互作用的核苷酸类似物的倒数平均寿命比宏观数据预测的大一到几倍。动力学和平衡数据的核苷酸（acto）肌球蛋白的相互作用来自单分子显微镜现在有一个生化和生理的框架。这对于马达蛋白的单分子力学研究是重要的。
Single-molecule and macroscopic reactions of fluorescent nucleotides with myosin have been compared. The single-molecule studies serve as paradigms for enzyme-catalyzed reactions and ligand-receptor interactions analyzed as individual stochastic processes. Fluorescent nucleotides, called Cy3-EDA-ATP and Cy5-EDA-ATP, were derived by coupling the dyes Cy3.29.OH and Cy5.29.OH (compounds XI and XIV, respectively, in Mujumdar et al. (1993, Bioconjug. Chem. 4:105-111)) with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP (EDA-ATP). The ATP(ADP) analogs were separated into their respective 2'- and 3'-O-isomers, the interconversion rate of which was 30[OH-] s(-1) (0.016 h(-1) at pH 7.1) at 22 degrees C. Macroscopic studies showed that 2'(3')-O-substituted nucleotides had properties similar to those of ATP and ADP in their interactions with myosin, actomyosin, and muscle fibers, although the ATP analogs did not relax muscle as well as ATP did. Significant differences in the fluorescence intensity of Cy3-nucleotide 2'- and 3'-O-isomers in free solution and when they interacted with myosin were evident. Single-molecule studies using total internal reflection fluorescence microscopy showed that reciprocal mean lifetimes of the nucleotide analogs interacting with myosin filaments were one- to severalfold greater than predicted from macroscopic data. Kinetic and equilibrium data of nucleotide-(acto)myosin interactions derived from single-molecule microscopy now have a biochemical and physiological framework. This is important for single-molecule mechanical studies of motor proteins.