Pulse‐Fluorometry Study on Actin and Heavy Meromyosin Using F‐Actin Labelled with N‐(1‐Pyrene)maleimide

Abstract

The single‐photoelectron counting technique was used for measurement of the fluorescence decay kinetics of N‐(1‐pyrene)maleimide conjugated to the fast reactive cysteine of actin. The fluorescence decay curve of the labelled G‐actin could not be described by a single‐exponential function but by a double‐exponential function. Polymerization of actin was accompanied by significant changes in the decay parameters of the two decay components. We found that the ionic strength, which plays an important role in the G‐F equilibrium, scarcely affected these parameters provided that the labelled actin exists in the monomeric state. Thus it is suggested that the conformational change of actin protomer occurs at the time of association. When heavy meromyosin was added to the labelled F‐actin, the decay parameter changed monotonically on increasing saturation of binding of heavy meromyosin and it levelled‐off around a ratio of heavy meromyosin:actin of 0.5 mol/mol. Decay parameters under the influence of heavy meromyosin had values intermediate between those observed for the labelled G‐actin and for the labelled F‐actin. Therefore, it is suggested that binding of heavy meromyosin to F‐actin alters the conformation of actin protomer towards that similar to G‐actin.

Fesselin, a Synaptopodin-like Protein, Stimulates Actin Nucleation and Polymerization

“…In all cases, the excitation wavelength (λ x ) 1 was set with a monochromator, and the emission wavelength (λ m ) was set either with a monochromator (conventional fluorometer) or with long-pass filters (stopped-flow). The following excitation and emission wavelengths were used to monitor changes in pyrene fluorescence: λ x = 344 nm, λ m = 366 nm (12); λ x = 365 nm, λ m = 385 nm (13); and λ x = 365 nm, λ m = 407 nm (15,20). Each method gave the same rate of polymerization although the amplitude of the signal and the extent to which scattered light contributed to the final signal varied with each case.…”

“…Pyrene-labeled actin was prepared by reacting F-actin with N- (1-pyrenyl)iodoacetamide (Molecular Probes) using the method of Kouyama and Mihashi (15). The actin was depolymerized by exhaustive dialysis, clarified by centrifugation, and then purified by gel-filtration chromatography on a Sephacryl S-200 column.…”

“…The actin was depolymerized by exhaustive dialysis, clarified by centrifugation, and then purified by gel-filtration chromatography on a Sephacryl S-200 column. The actin concentration was determined by absorbance measurements at 290 nm corrected for light scattering at 500 nm using the extinction coefficient of 0.617 mg −1 mL cm −1 (15). The pyrene-actin concentration and the labeling extent were calculated from the absorbance at 290 and 344 nm (16).…”

An EPR study of the rotational dynamics of actins from striated and smooth muscle and their complexes with heavy meromyosin

“…Signals that are sensitive to local conformational changes were usually reported to give a unidirectional response which was a linear function of the fraction of actin monomers bound [15,31,62,[72][73][74], although cooperative (non-linear) effects were also reported [6, 611 and other authors observed a biphasic change in ultraviolet absorption [75] and fluorescence intensity of static fluorescence anisotropy of etheno-ADP in F-actin [31,76]. Probes reporting the rotational mobility of F-actin give a cooperative response [4,6,11,12,15,771 (and this work), usually unidirectional, but a biphasic response was observed in the quasi-elastic light-scattering [4, 61.…”

Molecular Basis for Barbed End Uncapping by CARMIL Homology Domain 3 of Mouse CARMIL-1

“…Actin was purified from rabbit skeletal muscle as described previously (39,40). Monomeric actin was purified by gel filtration chromatography using S-300 resin to remove oligomers.…”