Interaction of myosin.ADP.fluorometal complexes with fluorescent probes and direct observation using quick-freeze deep-etch electron microscopy.
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.
Department of Bioengineering, Soka University, Hachioji, Tokyo 192-8577. shinsaku@t.soka.ac.jp
Shinsaku
S
Maruta
Yasuo
Y
Uyehara
Tomoki
T
Aihara
Eisaku
E
Katayama
D000244:Adenosine Diphosphate / Q000737:chemistry*; D017607:Aluminum Compounds / Q000737:chemistry; D001608:Beryllium / Q000737:chemistry; D005456:Fluorescent Dyes; D005459:Fluorides / Q000737:chemistry; D005613:Freeze Etching; D008854:Microscopy, Electron; D009218:Myosins / Q000648:ultrastructure*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D017607:Aluminum Compounds; D005456:Fluorescent Dyes; D000244:Adenosine Diphosphate; D009218:Myosins; D001608:Beryllium; D005459:Fluorides; C032311:aluminum fluoride