The recovery of the polymerizability of Lys-61-labelled actin by the addition of phalloidin. Fluorescence polarization and resonance-energy-transfer measurements

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Abstract
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°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.