Satoru Fujime

Dynamic study of F-actin by quasielastic scattering of laser light

Abstract A newly developing technique, quasielastic scattering of laser light, was applied to the dynamic study of F-actin interacting with other muscle proteins, heavy meromyosin, S-1 (i.e. a single head of myosin) and tropomyosin. The following three systems were studied: (1) the interaction between F-actin and heavy meromyosin or S-1 in the absence of ATP; (2) the interaction between F-actin and tropomyosin at various salt concentrations; and (3) the interaction between heavy meromyosin and the complex of F-actin and tropomyosin in the absence of ATP. F-actin was most flexible when the mole ratio of heavy meromyosin to F-actin monomer was 1:6. At this ratio, one heavy meromyosin molecule binds to about half a pitch of one strand of the actin filament. On the other hand, flexibility of F-actin was not changed on the addition of S-l. The addition of tropomyosin to F-actin solutions made F-actin stiffer. Tropomyosin appears to bind directly to F-actin up to a weight ratio of tropomyosin to F-actin of 1:6. This direct binding of tropomyosin affected the interaction between F-actin and heavy meromyosin. On the addition of heavy meromyosin to the complex solution of F-actin and tropomyosin, the complex was most flexible when the molar ratio of heavy meromyosin to F-actin was between 1:3 and 1:2. F-actin is not a rigid rod; rather, it appears to be a partially flexible chain. The flexibility of F-actin may have an important role in the molecular mechanism of muscular contraction.

Effect of calcium ions on the flexibility of reconstituted thin filaments of muscle studied by quasielastic scattering of laser light

Abstract Quasielastic scattering of laser light was used to examine the effect of calcium ions on the flexibility of a reconstituted thin filament, namely, an F-actin/tropomyosin/troponin complex of striated muscle. The results showed that the flexibility of a reconstituted thin filament changed reversibly at about 1 μ m of free calcium ions (i.e. the physiological concentration). Below 1 μ m -Ca 2+ a tropomyosin/troponin system suppressed the bending motion of F-actin. This suppression was removed by calcium ions above 1 μ m , and the F-actin flexibility became almost the same as that of an F-actin/tropomyosin complex. A quantitative study showed that calcium ions affected the reconstituted thin filament in two ways at the free calcium ion concentrations of about 1 μ m and 20 μ m , corresponding, respectively, to the two different calcium binding constants of troponin. An over-all conformational change of a reconstituted thin filament is brought about by calcium ions. It is suggested that the effect of calcium ions on troponin can be transferred to F-actin by altering the binding between tropomyosin and several monomer units in F-actin. The possible role of the dynamic nature of F-actin in muscle contraction is discussed.

Quasi-Elastic Light Scattering from Solutions of Macromolecules. II. Doppler Broadening of Light Scattered from Solutions of Semi-Flexible Polymers, F-Actin

By use of an optical self-beat method, the spectral density of light scattered from solutions of a semi-flexible polymer, F-actin, was measured for various scattering angles. The analysis of spectral densities gave the relaxation time of the lowest order internal mode of a bending motion of F-actin. From the experimental value of the relaxation time, the mean square end-to-end distance was estimated which agrees with the observation by electron microscopy. The present method makes it possible to estimate the flexibility of polymers.

Flexural rigidity of bacterial flagella studied by quasielastic scattering of laser light

Bacterial flagella are organelles of locomotion. Since their thickness is less than 200 A, it is impossible to observe individual intact flagella by light microscopy. In order to estimate the flexural rigidity of flagella in vitro, reconstituted flagella of Salmonella were studied by quasielastic scattering of laser light. The main findings were as follows. The bacterial flagellum is very stiff. Its flexural rigidity is of the order of 10−15 dyncm2 for straight-type flagella. Among mutant strains, the normal-type flagellum seems to be the most stiff. Curly- and straight-type flagella follow in that order. The mean length of reconstituted flagella is nearly equal irrespective of widely varied weight ratios of flagellin to seed at the reconstitution, provided that the ratio exceeds some critical value. This suggests that there exists some self-control mechanism in the polymerization process of flagellin.

Quasi-Elastic Light Scattering from Solutions of Macromolecules. I. Doppler Broadening of Light Scattered from Solutions of Tobacco Mosaic Virus Particles

By use of a laser light source and photoelectric light mixing technique, spectral densities of light scattered from solutions of tobacco mosaic virus particles were measured in order to obtain translational and rotational diffusion coefficients, D and \(\varTheta\), of the particle. The observed values of D 20 and \(\varTheta_{20}\) are 0.29±0.01×10 -7 cm 2 sec -1 and 360±20 sec -1 , respectively, for the 3 mg/ml solution. These coefficients were markedly dependent on the protein concentration of solutions below 1 mg/ml. In addition, the intensity due to the coupling of translational and rotational Brownian motions was observed, which agreed with a theoretical prediction.

Dynamic light scattering from dilute suspensions of thin discs and thin rods as limiting forms of cylinder, ellipsoid and ellipsoidal shell of revolution.

A previous formulation of the field correlation function G1(tau) of light quasielastically scattered from suspensions of rigid rods undergoing anisotropic translational as well as rotational diffusion (T. Maeda and S. Fujime, Macromolecules 17 (1984) 1157) was extended to the cases of suspensions of cylinders (length L and radius R), ellipsoids and ellipsoidal shells of revolution (x2/b2 + y2/b2 + z2/a2 = 1). The present formulation includes that for suspensions of rigid rods in the limit of KR 1 or in the limit of b/a 1 and Kb 1 (an extremely prolate ellipsoid), and also that for suspensions of discs in the limit of KL 1 or in the limit of b/a 1 and Ka 1 (an extremely oblate ellipsoid), where K is the length of the scattering vector. Explicit forms of G1(tau), of the first cumulant Gamma of G1(tau) and of the dynamic form factors will be given, and numerical methods suitable for computation of dynamic form factors will be discussed. The present results can be applied to the analysis of experimental data for dilute suspensions of thin rods and thin discs. When the situation is favorable, our method can provide transport coefficients D1, D3, and Theta from dynamic light-scattering data only, where D(1) and D(3) are, respectively, the translational diffusion coefficients parallel with the x (y) and z axes, and Theta the rotational diffusion coefficient around the x (y) axis.

Optical diffraction study of muscle fibers: I. A theoretical basis

Abstract We present a simple theoretical model of optical diffraction by striated muscle fibers. The model accounts for (1) changes in diffraction intensity during isometric contraction, (2) spectra of quasielastically scattered light from isometrically contracting muscle, and (3) an electro-optical effect or intensity modulation of diffraction lines by applied electric field. Items (1) and (2) are concerned with the fluctuations of the sarcomere structure during isometric contraction, and item (3) is concerned with the lateral motions of, or the flexibility of, thin filaments in striated muscle at rest.

Optical diffraction study of muscle fibers: II. Electro-optical properties of muscle fibers

When an electric field is applied along the fiber axis, the intensities of all observable optical diffraction lines of skeletal muscle fibers increase. This electro-optical effect was extensively studied and it was confirmed that the effect is due to the interaction between electric dipole moments of thin filaments and the applied field. From the present study on the intensity modulation due to applied field in sinusoidal and square forms, we confirmed that (1) the thin filament is a semiflexible rod, (2) the second order mode of the bending motion of thin filaments contributes to the electro-optical effect of muscle fibers at higher frequencies of a sinusodidal field or shorter durations of a square field, (3) the induced moment has no appreciable effect, and (4) the estimated value of the flexural rigidity of thin filaments strongly depends on the concentrations of free calcium ions in the myofibrillar space.

Quasielastic Light Scattering under Optical Microscope

A system for quasielastic light scattering under an optical microscope is proposed. This system makes it possible to obtain some information about dynamic properties of macromolecules in a limited part (100×100 μ2) of a living cell.

Dynamic light-scattering study of muscle F-actin. II

By dynamic light scattering, the intensity autocorrelation function, G2(tau) = B[1 + beta[g1(tau)[2], was obtained over the scattering angles (theta) from 30 to 130 degrees in steps of 10 degrees for semidilute solutions of muscle F-actin and of F-actin complexed with heavy meromyosin in the absence of ATP (acto-HMM), where B is the baseline and beta a constant. The main findings were: (1) A 0.5 mg/ml F-actin solution gave nonreproducible spectra at theta less than or equal to 40 degrees but quite reproducible spectra at theta greater than or equal to 50 degrees, with beta = 0.9-0.8 at all theta values. Nonreproducibility of spectra at low theta values was concluded to be due to restricted motions of very long filaments confined in cages or zig-zag tubing formed by a major fraction of filaments, where the very long filaments were those at a distant tail of an exponential length distribution and the major fraction of filaments were those with lengths around Ln-2Ln, Ln being the number-average length. Spectral widths were compared with theoretical ones for rigid rods averaged over the length distribution with Ln = 900 nm, and were suggested to be largely contributed at high theta values from bending motions of filaments. (2) Acto-HMM solutions at 0.5 mg/ml F-actin and at weight ratios of HMM to F-actin of 0.5-2 gave spectra which, with respect to theta, behaved very similarly to those of F-actin alone. The spectral widths, however, drastically decreased with the weight ratio up to unity and stayed virtually constant above unity. In contrast to a previous study (F.D. Carlson and A.B. Fraser, J. Mol. Biol. 89 (1974) 273), beta values of acto-HMM were as large as those of F-actin alone. Acto-HMM was concluded to travel a distance far greater than 1/K with a mobility smaller than that of F-actin, where K = (4 pi/lambda) sin(theta/2), lambda being the wavelength of light in the medium. These results suggest that acto-HMM gels are very soft even though they did not pour from an inverted cell. Based on several intuitive models which give a mutual relationship between the beta value and modes of motion of scatterers, we discuss the restricted motions responsible for nonreproducibility of spectra at low angles and large beta values of acto-HMM gels at all theta values and weight ratios so far studied.