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

Abstract

Modification of Lys-61 in actin with fluorescein-5-isothiocyanate (FITC) blocks actin polymerization [Burtnick, L. D. (1984) Biochim. Biophys. Acta 791, [57][58][59][60][61][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-I 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-I and the acceptor fluorescein-5-isothiocyanate bound to Lys-61 of actin in the rigor complex was measured. The transfer efficiency was 0.39 f 0.05 which corresponds to the distance of 5.2 f 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.Bovine pancreatic DNase I depolymerizes F-actin and forms a 1 : l complex [I] with actin monomers. The threedimensional structure of the complex of actin and DNase I has been obtained by X-ray crystallography at 0.45-nm resolution [2]. According to this model, an actin subunit consists of a small and a large domain with the dimensions of 6.7 x 4.0 x 3.7 nm, the small domain containing the N-terminus. The phosphate of a bound nucleotide is located between the two domains. DNase I binds mainly to a loop region in the small domain of actin. On the other hand, chemical cross-linking methods revealed that residues at positions 50, 53, 61, 68 and/or 69 are within 0.3 nm of DNase I binding site [3]. These residues may be located near actin-actin binding site, since the chemical modifications of , Lys-61 [5] and impairs the polymerizability of actin without a gross conformational change such as detected by circular dichroic measurements.Phalloidin binds tightly to F-actin but not to G-actin [7, 81 and stabilizes the filamentous structure of actin not only towards depolymerizing conditions or agents but also towards heat denaturation and proteolytic degradation [9 -1 I]. The binding of phalloidin influences the polymerization of actin by stabilizing nuclei and polymers as they are formed [8] and also induces a conformational change in actin [12].The interaction of actin and myosin i...

Spatial relationship between the nucleotide‐binding site, Lys‐61 and Cys‐374 in actin and a conformational change induced by myosin subfragment‐1 binding

“…In order to obtain additional information about the spatial relationship between labelled sites, the distances between Lys-61 and the nucleotide binding site or Cys-374 were measured. Recently, it has been demonstrated that FITC-actin recovers its polymerizability to form the same helical structure as normal F-actin in the presence of phalloidin [9]. This enabled the inter-monomer distance between FITC attached to Lys-61 in an actin monomer and 1,5-IAEDANS attached to Cys-374 in its nearest neighbour monomer to be measured.…”

“…(The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) On the other hand, when doubly-labelled actin with 1,SIAEDANS at Cys-374 and FITC at Lys-61 was polymerized in the presence of a twofold molar excess of phalloidin [Miki, M. (1987) Eur. J. Biochem.…”

The effect of the replacement of ADP with a photoaffinity ATP analogue, 2‐azido‐ADP, in F‐actin on its function

“…Actin, Sl, tropomyosin and troponin were prepared as described previously [6]. Synthesis of 2-azido-ATP was carried out according to the method of Czarnecki [7].…”

“…Absorption was measured with a Philips PUS800 and fluorescence spectra were measured with an SLM 8000. ATPase activity was measured using a radiometer pH-stat as previously reported [6]. Viscosity was measured at 25°C with an Ostwald viscometer having an outflow time of 52 s for water.…”

“…Viscosity was measured at 25°C with an Ostwald viscometer having an outflow time of 52 s for water. Protein concentrations were determined from the absorption as described previously [6] and F-actin concentration was determined using the Biuret method. …”