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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...
Ras activation of phosphoinositide 3-kinase (PI3K) is important for survival of transformed cells. We find that PI3Kγ is strongly and directly activated by H-Ras G12V in vivo or by GTPγS-loaded H-Ras in vitro. We have determined a crystal structure of a PI3Kγ/Ras.GMPPNP complex. A critical loop in the Ras binding domain positions Ras so that it uses its switch I and switch II regions to bind PI3Kγ...
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,...
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