The 2'-O- and 3'-O-Cy3-EDA-ATP(ADP) complexes with myosin subfragment-1 are spectroscopically distinct.
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 approximately 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.
Oiwa
Kazuhiro
K
Kanasi Advanced Research Center, Kobe 651-2492, Japan.
Jameson
David M
DM
Croney
John C
JC
Davis
Colin T
CT
Eccleston
John F
JF
Anson
Michael
M
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry*; D016880:Anisotropy; D005454:Fluorescence Polarization / Q000379:methods*; D007536:Isomerism; D046911:Macromolecular Substances; D008968:Molecular Conformation; D009038:Motion; D015879:Myosin Subfragments / Q000737:chemistry*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods*
D046911:Macromolecular Substances; D015879:Myosin Subfragments; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate