Fluorescence energy transfer measurements between the nucleotide binding site and Cys-373 in actin and their application to the kinetics of actin polymerization.
Intramonomer fluorescence energy transfer between the donor epsilon-ATP bound to the nucleotide-binding site and the acceptor 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole bound to Cys-373 in G-actin was measured by steady-state fluorimetry. Assuming for the orientation factor its dynamic limit K2 = 2/3, the donor and acceptor distance in a G-actin molecule was calculated to be about 3 nm. The intermonomer energy transfer in F-actin occurring between the donor bound to an actin monomer and the acceptor bound to the nearest-neighbour actin monomer was also measured and the distance was calculated to be about 4 nm. The kinetics of the actin polymerization process was studied by following the decrease in fluorescence intensity upon addition of salts to G-actin solution. The initial velocity of the fluorescence intensity change was proportional to the square of the initial G-actin concentration. The temperature dependence of the velocity was proportional to the square of the initial G-actin concentration. The temperature dependence of the velocity was proportional to exp(-10/RT). These results indicated that the initial fluorescence intensity change corresponds to monomer-dimer transformation and its activation enthalpy was 10 kcal/mol.
D009327:4-Chloro-7-nitrobenzofurazan; D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003545:Cysteine; D004735:Energy Transfer; D007700:Kinetics; D011108:Polymers; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000199:Actins; D011108:Polymers; D000255:Adenosine Triphosphate; D009327:4-Chloro-7-nitrobenzofurazan; D003545:Cysteine