| Budget Amount *help |
¥8,200,000 (Direct Cost: ¥8,200,000)
Fiscal Year 1999: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1998: ¥6,100,000 (Direct Cost: ¥6,100,000)
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| Research Abstract |
To obtain a consistent view of the nucleotide-induced conformational changes around the hydrophobic region near CysィイD1707ィエD1(SH1) in skeletal myosin subfragnent-1 (S-1), the two thiols SH1 and CysィイD1697ィエD1 (SH2) were labeled with the same environmentally sensitive fluorophore, 6-acyl-2-dimethylaminonaphthalene group, using 6-acryloyl-2-dimethylaminonaphthalene (acrylo-dan, AD) and 6-bromoacetyl-2-dimethylaminonaphthalene (BD), respectively. The resultant fluorescent derivatives, AD-S-1 and BD-S-1, have the same fluorophore at either SH2 or SH1, which was verified by inspections of changes in the ATPases and the localization of fluorescence after tryptic digestion and CNBr cleavage for the two derivatives. Especially, AD was found to be a very useful fluorescent reagent that readily reacts with only SH2 of S-1. Measurements of the nucleotide-induced changes in fluorescence emission spectra of AD-S-1 and BD-S-1 suggested that during ATP hydrolysis the environment around the fluorophore at SH2 is very distinct from that around the fluorophore at SH1, being defined as that the former has the hydrophobic and closed characteristics, whereas the latter has the hydrophilic and open ones. The K1 quenching study of the fluorescence of the two S-1 derivatives confirmed these results. The most straightforward interpretation for the present results is that during ATP hydrolysis, the helix containing SH2 is buried in hydrophobic side chains and rather reinforced, whereas the adjacent helix containing SH1 moves away from its stabilizing tertiary structural environment. The results suggest that the hydrophobic region near SH1 plays an important role in the motor function of myosin head.
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