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Biophys J, June 2000, p. 3048-3071, Vol. 78, No. 6
*Kansai Advanced Research Center, Communications Research
Laboratory, Kobe 651-2492, Japan, and National Institute
for Medical Research, London NW7 1AA, United Kingdom
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
] s1 (0.016 h1 at
pH 7.1) at 22°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.
Biophys J, June 2000, p. 3048-3071, Vol. 78, No. 6
© 2000 by the Biophysical Society 0006-3495/00/06/3048/24 $2.00
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