Spectroscopic and functional characterization of an environmentally sensitive fluorescent actin conjugate.
Rabbit skeletal muscle F-actin has been selectively labeled at a cysteine residue with the environmentally sensitive fluorophore 6-acryloyl-2-(dimethylamino)naphthalene. The fluorescent actin conjugate behaves similarly to native actin with respect to the polymerization kinetics, critical monomer concentration, and ability to form F-actin paracrystals. Upon polymerization to F-actin, the absorption of the actin conjugate is red-shifted, whereas the fluorescence emission is blue-shifted 740 wavenumbers and is accompanied by a decrease in the fluorescence bandwidth of 470 wavenumbers. These large shifts in the spectral properties of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) in actin provide a simple method for obtaining a spectral discrimination between the G- and F-actin populations during the polymerization reaction. Steady-state fluorescence techniques were used to study the environment of the fluorophore in the monomeric and polymeric forms of actin. Fluorescence emission spectral analysis and quenching and polarization studies of G-actin-Prodan indicated that the fluorophore lies immobile on the protein surface but with one of its faces in full contact with the solvent. In F-actin, the fluorophore has a limited exposure to the solvent and is located in a dielectric environment similar to those seen for Prodan in polar, aprotic solvents or buried within a protein matrix [Macgregor, R. B., Jr., & Weber, G. (1986) Nature (London) 318, 70-73]. Additionally, our results demonstrate that the Prodan molecule conjugated to F-actin is completely immobile during its fluorescence lifetime, exhibits an increase in the resonance energy transfer (RET) from tryptophan residues compared to that observed in G-actin, and shows evidence of homologous RET within the polymer.
Marriott
G
G
Abteilungen Molekulare Biologie und Biochemie, Max-Planck-Institut für Biophysikalische Chemie, Göttingen, FRG.
Zechel
K
K
Jovin
T M
TM
D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D005456:Fluorescent Dyes; D007700:Kinetics; D009132:Muscles / Q000378:metabolism; D009281:Naphthalenes / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D000199:Actins; D005456:Fluorescent Dyes; D009281:Naphthalenes; C038518:acrylodan; D015081:2-Naphthylamine