Anisotropy Decay of Labelled Actin

Abstract

G actin, labelled presumably on cysteine-373 with the fluorescent chromophore N-iodoacetyl-N'-(5 sulfo-1 -naphthyl)-ethylenediamine and purified by Sephacryl S-200 gel chromatography, migrated in one band on polyacrylamide gel electrophoresis and had the same polymerizability as unlabelled purified G actin. Anisotropy decays of labelled actin solutions have been studied at different ionic strengths and protein concentrations.It was found that these anisotropy decays could be fitted by a sum' of two exponential functions. Under low ionic strength or below the critical concentrations the longer correlation time (45 ns at 3.5 " C ) was independent of protein concentration and ionic strength. Above the critical concentration, the longer correlation time increased with ionic strength and protein concentration. In order to take into account that, under these conditions, the solutions contained a mixture of F and G actin at the critical concentration, the anisotropy decays were analysed as a sum of three exponential functions in which the longest correlation time characterized F actin. Since F actin correlation time also depended on actin concentration, an analysis with a sum of four exponential functions was performed, in which two fixed correlation times (100 ns and 900 ns at 3.5 "C) were introduced in order to characterize the F actin motions. The longer of these correlation times was attributed to regions where two actin filaments interact side by side, while the shorter one was attributed to filament regions free from intermolecular interactions.The small value of the free F actin correlation time indicates that the protomer peptide chain is very flexible around its C terminus, probably involving the motion of a molecular lobe. This flexibility might be an important factor in the interaction of actin with myosin during the muscular contraction.During muscular contraction, the globular heads of the myosin molecules which constitute the thick filament, interact cyclically with the actin protomers in the thin filaments [l].The thin filament also includes troponin and tropomyosin, the regulatory proteins which control the myosin-actin interaction, according to the calcium ion concentration in the cell [2]. Tropomyosin has been shown to lie along the grooves of the double-helix structure formed by the actin protomers in the filament. According to X-ray diffraction experiments, its position differs in the relaxed and the contracting states of the muscle [3]. The regulatory mechanism associated with this motion of tropomyosin has been explained by the steric blocking model [4], in which actin does not play an active role.Actin can be extracted from muscles with a lowionic-strength solvent in which this protein is dispersed as monomer molecules (G actin). If one increases the ionic strength, actin polymerizes in helical aggregates (F actin) [4]. F actin forms complexes with tropomyosin and troponin in which the arrangement of these molecules is the same as in the thin muscle filaments. Studies of these reconstituted filam...

Conformational Changes in Actin Filaments Induced by Formin Binding to the Barbed End

“…Transient absorption anisotropy experiments showed that the actin filaments are more flexible in twisting motion than in bending (42) and the correlation times are in the microsecond range. We agree therefore with previous interpretations (32,35,36) that the correlation times of a few hundred nanoseconds in fluorescence anisotropy decay experiments are attributed to restricted segmental motion within the actin filaments. The segment characterized by these correlation times can be an actin protomer performing restricted motion in the filament, or a set of a few neighboring protomers with restricted and concerted motion.…”

“…3 A). Previously it was established that the value of the longer correlation time is inversely proportional to the flexibility of the actin filaments (32,(34)(35)(36)(37). Therefore, the formin-induced decrease of the rotational correlation time suggested that the interactions between neighboring protomers became weaker, and the actin filaments became more flexible, due to the binding of the mDia1-FH2 (see Discussion).…”

Intermonomer flexibility of Ca‐ and Mg‐actin filaments at different pH values

“…These long rotational correlation times cannot be specific for the overall motion of the actin filaments, which occur on much longer time‐scale [55] and probably characterize the restricted motion of segments within the actin filaments. In such cases the value of the longer rotational correlation time resolved in fluorescence anisotropy decay experiments is inversely proportional to the flexibility of the actin filaments [12,34,35,56,57]. Accordingly, these data show that in the case of the Mg‐actin filaments the protein matrix is more rigid at pH 6.5 than at pH 7.4.…”

“…A large number of experiments confirm that the actin filaments can change their dynamic and/or conformational properties due to the effect of the altered physico‐chemical properties of the surroundings or to the effects of other proteins involved in the force generation [2–7]. The actin filaments present in different dynamic states [6,8–12] can have an effect on the function of the acto–myosin complex through the altered flexibility of the protein matrix [7,12], which can also have significant functional effects at the cellular level [6]. The better understanding of the relationship between the flexibility and the function of actin filaments seems to be important for describing the molecular details of the muscle contraction under physiological and pathological conditions.…”

Conformational dynamics of actin: Effectors and implications for biological function

“…Correlation times on the fs range reflect the rearrangements of atoms/molecules, and these structural changes can be resolved by X-ray crystallography, electron microscopy (EM), cryo-EM and femtobiological approaches [Egelman, 2000; Resch et al, 2002; Sundstrom, 2008]. The ns correlation times are related to the change in the restricted segmental motion of a monomer/protomer or a few neighbouring protomers and can be determined by time-dependent fluorescence anisotropy [Ikkai et al, 1979; Miki et al, 1982a, b] or conventional electron paramagnetic resonance (EPR) [Thomas et al, 1979; Mossakowska et al, 1988]. The torsional twisting and bending motions of the whole actin filament characterised by correlation times in the μs and μs–ms range, can be described by phosphorescence anisotropy [Prochniewicz et al, 1996a; Yoshimura et al, 1984], saturation transfer (ST) EPR [Thomas et al, 1979; Hegyi et al, 1988], and transient absorption anisotropy measurements [Mihashi et al, 1983].…”