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 changes 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-dimethylaminonaphthalene (acrylodan, 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 KI 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.
Hiratsuka
T
T
Department of Chemistry, Asahikawa Medical College, Asahikawa, Hokkaido 078-8510, Japan. toshiaki@asahikawa-med.ac.jp
D015081:2-Naphthylamine / Q000031:analogs & derivatives; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D003488:Cyanogen Bromide; D003545:Cysteine / Q000737:chemistry*; D005456:Fluorescent Dyes; D007700:Kinetics; D015394:Molecular Structure; D009124:Muscle Proteins / Q000737:chemistry; D018482:Muscle, Skeletal; D015879:Myosin Subfragments / Q000737:chemistry*; D009711:Nucleotides / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014357:Trypsin; D018909:src Homology Domains
C122155:6-bromoacetyl-2-dimethylaminonaphthalene; D005456:Fluorescent Dyes; D009124:Muscle Proteins; D015879:Myosin Subfragments; D009711:Nucleotides; D013438:Sulfhydryl Compounds; C038517:prodan; C038518:acrylodan; D000255:Adenosine Triphosphate; D015081:2-Naphthylamine; D014357:Trypsin; D003545:Cysteine; D003488:Cyanogen Bromide