Behavior of Cys-707 (SH1) in myosin associated with ATP hydrolysis revealed with a fluorescent probe linked directly to the sulfur atom.
4-Fluoro-7-sulfamoylbenzofurazan (ABDF) has a rather small fluorophore that is linked directly to the sulfur atom of thiols without a flexible alkyl chain (Imai, K., and Toyo'oka, T. (1987) Methods Enzymol. 143, 67-75). In the present study I examined the fluorescent and chemical properties of ABDF as an environmentally sensitive probe for Cys-707 (SH1) of myosin subfragment-1 (S-1) to monitor the behavior of SH1 associated with ATP hydrolysis. ABDF was very stable to long irradiation and nonfluorescent before attachment to thiols, permitting the continuous monitoring of the labeling reaction. The fluorophore was useful as an environmentally sensitive probe for thiol groups in proteins. SH1 of S-1 was specifically labeled with ABDF. When ATP, adenyl-5'-yl imidodiphosphate, and ADP were added to the labeled S-1 (ABD-S-1), the fluorescence intensity at 500 nm increased by 110, 66, and 53%, respectively. Binding of actin to ABD-S-1 resulted in a decrease in the fluorescence by 30%. The fluorophore attached to SH1 was found to be located in a more hydrophobic environment in the presence of ATP than in the absence of ligand. KI fluorescence quenching studies suggested that the binding of ATP causes a movement of SH1 toward a more hydrophobic protein interior, whereas it goes back to the opposite direction after ATP hydrolysis. Thus, ABDF is very useful as an environmentally sensitive fluorescent probe for SH1 that monitors ligand-induced changes in the behavior of the thiol.
Hiratsuka
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Department of Chemistry, Asahikawa Medical College, Hokkaido, Japan.
D020106:Acrylamide; D000178:Acrylamides / Q000737:chemistry; D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D003545:Cysteine / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006868:Hydrolysis; D009218:Myosins / Q000378:metabolism*; D009282:Naphthalenesulfonates / Q000737:chemistry; D009711:Nucleotides / Q000378:metabolism; D010069:Oxadiazoles / Q000737:chemistry*; D011193:Potassium Iodide / Q000737:chemistry; D011817:Rabbits; D013455:Sulfur / Q000737:chemistry*
D000178:Acrylamides; D000199:Actins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D009711:Nucleotides; D010069:Oxadiazoles; D011193:Potassium Iodide; D020106:Acrylamide; C048876:1,5-AEDANS; D013455:Sulfur; D000255:Adenosine Triphosphate; C046364:4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole; D009218:Myosins; D003545:Cysteine