K. Oiwa1, M. Anson2, J.F. Eccleston2, A. Yamada1, H. Nakayama1 & D.R. Trentham2
1Kansai Advanced Research Center, Kobe 651-24, Japan, 2National Institute for Medical Research, London NW7 1AA, U.K.
Using evanescent-wave laser excitation and fluorescence video microscopy, single molecules of fluorescent nucleotide analogs have been visualized, recorded and their life-times bound to myosin measured. We have used Cy3-EDA-ATP and Cy3-EDA-ADP, the conjugates of Cy3.29.OH with 2'(3')-O-(N-2-aminoethyl)carbamoyl-adenosine 5'-triphosphate and 5'-diphosphate, respectively to determine the kinetics of association and dissociation of individual molecules from rabbit fast-twitch skeletal myosin in vitro. Synthetic filaments on a quartz surface appeared as uniformly-bright fluorescent images with 5 nM Cy3-EDA-ATP or with 300 nM Cy3-EDA-ADP. At 50 pM Cy3-EDA-ATP or at 3 nM Cy3-EDA-ADP discrete bright spots were visible on the filaments. These appeared suddenly, remained at the same position and brightness for a while and then abruptly disappeared. The spot dwell times fitted a single exponential of rates 0.14 s-1 for Cy3-EDA-ATP at 23 °C and 0.43 s-1 for Cy3-EDA-ADP at 10 °C; close to the turnover rate, 0.03 s-1, of Cy3-EDA-ATP at 20 °C and dissociation rate-constant, 0.67 s-1, of Cy3-EDA-ADP from myosin S1 at 10 °C measured by stopped-flow. The corresponding association rate-constants were estimated as 2 x 106 M-1s-1 for Cy3-EDA-ATP and 1.8 ( 105 M-1s-1for Cy3-EDA-ADP). These results show that single-molecule events can be used to determine the kinetics of myosin-nucleotide interactions. These studies with Cy3-EDA-ADP serve as a model for ligand-receptor binding at the level of single molecules.