Microsecond Rotational Motions of Eosin-labeled Myosin Measured by Time-resolved Anisotropy of Absorption and Phosphorescence

Abstract

We have studied submicrosecond and microsecond rotational motions within the c>ontractile protein myosin by observing the time-resolved anisotropy of both absorption and emission from the long-lived triplet st,ate of eosin-5-iodoacetamide c*ovalently bound to a specific site on the myosin head. These results, reporting anisotropy data up to 50 microseconds after excitation. extend by two orders of magnitude the time range of data on time-resolved site-specific probe motion in myosin. Optical and enzymatic analyses of the labeled myosin and its chymotryptic digests show that more than 95 % of the probe is specifically attached to sulfhydryl-1 (SH,) on the myosin head. In a solution of labeled subfragment-l (S-l) at 4”C, absorption anisotropy at 0.1 ps after a laser pulse is about 0.27. This anisotropy decays exponentially with a rot,ational correlation time of 210 ns, in good agreement with the theoretical prediction for end-over-end tumbling of S-l, and with times determined previously by fluorescence and electron paramagnetic resonance. In ayueous glycerol solutions, this correlation time is proportional to viscosity/temperature in the microsecond time range