Distance between nucleotide site and cysteine-373 of G-actin by resonance energy transfer measurements.
The distance between the nucleotide site and the reactive cysteine-373 of G-actin was determined from resonance energy transfer measurements by using 1,N6-ethenoadenosine triphosphate (epsilon ATP) as the donor and 4-[N-(iodoacetoxy)ethyl N methyl]amino 7 nitrobenz 2 oxa 1,3 diazole covalently attached to the sulphydryl group as acceptor. The quenching of the lifetime of bound donor in the presence of attached acceptor arose predominantly from transfer of excitation energy. The polarization spectrum of free epilson ATP in glycerol revealed that the minimum value of its fundamental anisotropy is 0.32 at 340 nm, indicating that the maximum value of the angle between the absorption and emission dipoles of the ethenoadenosine moiety is 21 degrees. The polarization result indicates that the bound nucleotide is depolarized and has considerable motional freedom. This motion is restricted and unlikely to be rapid or isotropic during the time interval of energy transfer. The attached acceptor is highly immobile, however. The range of the donor-acceptor distance is 24-45 A. This range was not affected by polymerization. In the absence of independent structural information it is not possible to assign a single value to the donor-acceptor separation.
D000199:Actins / Q000032:analysis*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D003545:Cysteine / Q000032:analysis*; D004735:Energy Transfer; D004985:Ethenoadenosine Triphosphate / Q000032:analysis*; D010069:Oxadiazoles / Q000032:analysis; D013050:Spectrometry, Fluorescence
D000199:Actins; D004985:Ethenoadenosine Triphosphate; D010069:Oxadiazoles; C035573:4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole; D000255:Adenosine Triphosphate; D003545:Cysteine