ATP-induced opposite changes in the local environments around Cys(697) (SH2) and Cys(707) (SH1) of the myosin motor domain revealed by the prodan fluorescence

To obtain a consistent view of the nucleotide-induced conformational change s around Cys(697) (SH2) and Cys(707) (SH1) in skeletal myosin subfragment-1 (S-1), the two thiols were labeled with the same environmentally sensitive fluorophore, 6-acyl-2-dimethylaminonaphthalene group, using 6-acryloyl-2-d imethylaminonaphthalene (acrylodan, AD) and 6-bromoacetyl-2-dimethylaminona phthalene (BD), respectively. The resultant fluorescent derivatives, AD-S-1 and BD-S-1, have the same fluorophore at either SH2 or SH1, which was veri fied by inspections of changes in the ATPases and the localization of fluor escence after tryptic digestion and CNBr cleavage for the two derivatives. Especially, AD was found to be a very useful fluorescent reagent that readi ly reacts with only SH2 of S-1. Measurements of the nucleotide-induced chan ges in fluorescence emission spectra of ADS-1 and BD-S-1 suggested that dur ing ATP hydrolysis the environment around the fluorophore at SH2 is very di stinct from that around the fluorophore at SH1, being defined as that the f ormer has the hydrophobic and closed characteristics, whereas the latter ha s the hydrophilic and open ones, The KI quenching study of the fluorescence of the two S-1 derivatives confirmed these results. The most straightforwa rd interpretation for the present results is that during ATP hydrolysis, th e helix containing SH2 is buried in hydrophohic side chains and rather rein forced, whereas the adjacent helix containing SH1 moves away from its stabi lizing tertiary structural environment.