The recovery of the polymerizability of Lys-61-labelled actin by the addition of phalloidin. Fluorescence polarization and resonance-energy-transfer measurements.
Modification of Lys-61 in actin with fluorescein-5-isothiocyanate (FITC) blocks actin polymerization [Burtnick, L. D. (1984) Biochim. Biophys. Acta 791, 57-62]. FITC-labelled actin recovered its ability to polymerize on addition of phalloidin. The polymers had the same characteristic helical thread-like structure as normal F-actin and the addition of myosin subfragment-1 to the polymers formed the characteristic arrowhead structure in electron microscopy. The polymers activated the ATPase activity of myosin subfragment-1 as efficiently as normal F-actin. These results indicate that Lys-61 is not directly involved in an actin-actin binding region nor in myosin binding site. From static fluorescence polarization measurements, the rotational relaxation time of FITC-labelled actin filaments was calculated to be 20 ns as the value reduced in water at 20 degrees C, while any rotational relaxation time of 1,5-IAEDANS bound to Cys-374 on F-actin in the presence of a twofold molar excess of phalloidin could not be detected by static polarization measurements under the same conditions. This indicates that the Lys-61 side chain is extremely mobile even in the filamentous structure. Fluorescence resonance energy transfer between the donor 1,5-IAEDANS bound to SH1 of myosin subfragment-1 and the acceptor fluorescein-5-isothiocyanate bound to Lys-61 of actin in the rigor complex was measured. The transfer efficiency was 0.39 +/- 0.05 which corresponds to the distance of 5.2 +/- 0.1 nm, assuming that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime and that the transfer occurs between a single donor and an acceptor.
D000199:Actins / Q000032:analysis*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D004735:Energy Transfer; D005454:Fluorescence Polarization; D008239:Lysine; D008433:Mathematics; D008854:Microscopy, Electron; D009282:Naphthalenesulfonates / Q000378:metabolism; D009842:Oligopeptides; D010590:Phalloidine; D011108:Polymers / Q000378:metabolism; D011817:Rabbits
D000199:Actins; D009282:Naphthalenesulfonates; D009842:Oligopeptides; D011108:Polymers; D010590:Phalloidine; C006421:1,5-I-AEDANS; D017301:Ca(2+) Mg(2+)-ATPase; D008239:Lysine