Spectral properties of fluorescent derivatives of the oligomycin sensitivity conferring protein and analysis of their interaction with the F1 and F0 sectors of the mitochondrial ATPase complex.
In order to study the kinetics and the nature of the interactions between the oligomycin sensitivity conferring protein (OSCP) and the F0 and F1 sectors of the mitochondrial ATPase complex, fluorescent derivatives of OSCP, which are fully biologically active, have been prepared by reaction of OSCP with the following fluorescent thiol reagents: 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan), 2-(4-maleimidylanilino)naphthalene-6-sulfonic acid (Mal-ANS), N-(1-pyrenyl)maleimide (Mal-pyrene), 7-(diethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin (Mal-coumarin), and fluorescein 5-maleimide (Mal-fluorescein). The preparation of these derivatives was based on the previous finding that the single cysteinyl residue of OSCP, Cys 118, can be covalently modified by alkylating reagents without loss of biological activity [Dupuis, A., Issartel, J. P., Lunardi, J., Satre, M., & Vignais, P. V. (1985) Biochemistry 24, 728-733]. For all fluorescent probes used, except Mal-pyrene and Mal-fluorescein, the emission spectra of conjugated OSCP were blue-shifted relative to those of the corresponding mercaptoethanol adducts, indicating that the fluorophores attached to Cys 118 were located in a hydrophobic pocket. These results were consistent with the high quantum yields and the increased fluorescence lifetimes of conjugated OSCP compared to mercaptoethanol adducts in aqueous buffer. They also fit with quenching data obtained with potassium iodide which showed that the fluorophore is shielded from the aqueous medium when it is attached to Cys 118 of OSCP. Especially noticeable was the wide half-width of the OSCP-acrylodan emission peak compared to that of mercaptoethanol-acrylodan.(ABSTRACT TRUNCATED AT 250 WORDS)
Duszynski
J
J
Department of Cellular Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
Dupuis
A
A
Lux
B
B
Vignais
P V
PV
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D005456:Fluorescent Dyes; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013367:Submitochondrial Particles / Q000201:enzymology
D002352:Carrier Proteins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D008565:Membrane Proteins; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; C020439:oligomycin sensitivity-conferring protein