J. Biochem, 2004, Vol. 136, No. 1 57-64
© 2004 The Japanese Biochemical Society
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Interaction of Myosin·ADP·Fluorometal Complexes with Fluorescent Probes and Direct Observation Using Quick-Freeze Deep-Etch Electron Microscopy
1 Department of Bioengineering, Soka University, Hachioji, Tokyo 192-8577; 2 Division Biomolecular Imaging, Institute of Medical Science, University of Tokyo, Minato-Ku, Tokyo 108-8639; and PREST, JSP, Kawaguchi, Saitama
Myosin forms stable ternary complexes with ADP and phosphate analogues of fluorometals that mimic different ATPase reaction intermediates corresponding to each step of the cross-bridge cycle. In the present study, we monitored the formation of ternary complexes of myosin·ADP·fluorometal using the fluorescence probe prodan. It has been reported that the fluorescence changes of the probe reflect the formation of intermediates in the ATPase reaction [Hiratsuka (1998) Biochemistry 37, 7167–7176]. Prodan bound to skeletal muscle heavy-mero-myosin (HMM)·ADP·fluorometal, with each complex showing different fluorescence spectra. Prodan bound to the HMM·ADP·BeFn complex showed a slightly smaller red-shift than other complexes in the presence of ATP, suggesting a difference in the localized conformation or a difference in the population of BeFn species of global shape. We also examined directly the global structure of the HMM·ADP·fluorometal complexes using quick-freeze deep-etch replica electron microscopy. The HMM heads in the absence of nucleotides were mostly straight and elongated. In contrast, the HMM heads of ternary complexes showed sharply kinked or rounded configurations as seen in the presence of ATP. This is the first report of the direct observation of myosin-ADP-fluorometal ternary complexes, and the results suggest that these complexes indeed mimic the shape of the myosin head during ATP hydrolysis.
* To whom correspondence should be addressed.:A/Prof. Shinsaku Maruta, Department of Bioengineering, Faculty of Engineering, Soka University, Phone: +81-426-91-9443, Fax: +81-426-91-9312, E-mail: shinsaku{at}t.soka.ac.jp