The 2′- O- and 3′- O-Cy3-EDA-ATP(ADP) Complexes with Myosin Subfragment-1 are Spectroscopically Distinct

Abstract

Ribose-modified highly-fluorescent sulfoindocyanine ATP and ADP analogs, 2′(3′)- O-Cy3-EDA-AT(D)P, with kinetics similar to AT(D)P, enable myosin and actomyosin ATPase enzymology with single substrate molecules. Stopped-flow studies recording both fluorescence and anisotropy during binding to skeletal muscle myosin subfragment-1 (S1) and subsequent single-turnover decay of steady-state intermediates showed that on complex formation, 2′- O- isomer fluorescence quenched by 5%, anisotropy increased from 0.208 to 0.357, and then decayed with turnover rate k cat 0.07 s −1; however, 3′- O- isomer fluorescence increased 77%, and anisotropy from 0.202 to 0.389, but k cat was 0.03 s −1. Cy3-EDA-ADP·S1 complexes with vanadate (V i) were studied kinetically and by time-resolved fluorometry as stable analogs of the steady-state intermediates. Upon formation of the 3′- O-Cy3-EDA-ADP·S1·V i complex fluorescence doubled and anisotropy increased to 0.372; for the 2′- O- isomer, anisotropy increased to 0.343 but fluorescence only 6%. Average fluorescent lifetimes of 2′- O- and 3′- O-Cy3-EDA-ADP·S1·V i complexes, 0.9 and 1.85 ns, compare with ∼0.7 ns for free analogs. Dynamic polarization shows rotational correlation times higher than 100 ns for both Cy3-EDA-ADP·S1·V i complexes, but the 2′- O-isomer only has also a 0.2-ns component. Thus, when bound, 3′- O-Cy3-EDA-ADP’s fluorescence is twofold brighter with motion more restricted and turnover slower than the 2′- O-isomer; these data are relevant for applications of these analogs in single molecule studies.