Koshin Mihashi

Torsional motion of eosin-labeled F-actin as detected in the time-resolved anisotropy decay of the probe in the sub-millisecond time range

The internal motion of F-actin in the time range from 10(-6) to 10(-3) second has been explored by measuring the transient absorption anisotropy of eosin-labeled F-actin using laser flash photolysis. The transient absorption anisotropy of eosin-F-actin at 20 degrees C has a component that decays in the submicrosecond time scale to an anisotropy of about 0.3. This anisotropy then decays with a relaxation time of about 450 microseconds to a residual anisotropy of about 0.1 after 2 ms. When the concentration of eosin-F-actin was varied in the range from 7 to 28 microM, the transient absorption anisotropy curves obtained were almost indistinguishable from each other. These results show that the anisotropy decay arises from internal motion of eosin-F-actin. Analysis of the transient absorption anisotropy curves indicates that the internal motion detected by the decay in anisotropy is primarily a twisting of actin protomers in the F-actin helix; bending of the actin filament makes a minor contribution only to the measured decay. The torsional rigidity calculated from the transient absorption anisotropy is 0.2 X 10(-17) dyn cm2 at 20 degrees C, which is about an order of magnitude smaller than the flexural rigidity determined from previous studies. Thus, we conclude that F-actin is more flexible in twisting than in bending. The calculated root-mean-square fluctuation of the torsional angle between adjacent actin protomers in the actin helix is about 4 degrees at 20 degrees C. We also found that the torsional rigidity is approximately constant in the temperature range from 5 to approximately 35 degrees C, and that the binding of phalloidin does not appreciably affect the torsional motion of F-actin.

On the polymerization of tropomyosin

Abstract The polymerization of tropomyosin was studied by light scattering, covering a wide range of protein and salt concentrations. Depolymerization of tropomyosin molecules with dilution was found at very dilute protein concentration, a phenomenon which had not been observed. Extrapolation to zero protein concentration gave the monomer molecular weight at every salt concentration, indicating complete depolymerization at infinite dilution. The linear relation between the molecular weight and the corresponding length of the molecules obtained from the angular dependence of scattered light, showed that tropomyosin molecules were polymerized linearly. A length per monomer molecular weight of polymerized molecules, however, did not give the monomer length, especially when taking account of polydispersity and different averages of molecular weight and rod length obtained by the light scattering measurements. The analysis of dissociation-association equilibrium was applied to the system, and this gave a conclusion that molecules were linearly aggregated including some extent of side-overlapping.

Nanosecond pulsefluorometry in polarized light of G-actin-ϵ-ATP and F-actin-ϵ-ADP

It has been recently found by Miki et al. [ 1 ] and Thames et al. [2] that ATP, the natural ligand of Gactin, can be replaced’by 1 ,i@ -ethenoadenosine triphosphate, E-ATP. In the present work, the anisotropy decay of the fluorescence of E-ATP bound to Gactin as well as of E-ADP bound to F-actin are investigated. From these studies, correlation times characterising the Brownian rotation of G-actin and flexibility of F-actin are obtained.

Interaction of actin water ϵ-ATP

Since Straub found actin, one of the major proteins of the contractile system [ 1 ] , the structural and functional relation of actin with bound nucleotide has been extensively studied. The essential role of the nucleotide in the biological activity of actin is however not yet clear [2,3]. As a new approach to this problem, we attempted to introduce E-ATP, a fluorescent analog of ATP [4], and found that G-actin binds e-ATP and the bound e-ATP is hydrolysed in the course of polymerization of actin in a very similar way to ATP. Structure and function of F-actin having bound E-ADP is almost the same as F-actin having bound ADP.

MOLECULAR CHARACTERISTICS OF G-ADP ACTIN.

Abstract G-ADP actin is prepared by rapid depolymerization of F-actin by sonication at low temperature. This G-ADP actin is identical with that prepared by Hayashi and Weber in its ability to polymerize without undergoing dephosphorylation. Studies on the physicochemical properties of G-ADP actin at low temperature show that it exists in a monomeric form, whose shape and size are similar to those of G-ATP actin; the average molecular weight of G-ADP actin determined by the Archibald method is 5.65 × 104. G-ADP actin has an intrinsic viscosity [η] = 0.10 deciliters per gram, partial specific volume V = 0.732 ml per gram , sedimentation constant S20,w0 = 3.38 (Svedberg units), and diffusion constant is D20,w0 = 5.28 × 10−7 cm2 per second. The effective hydrodynamic ellipsoid of G-ADP actin has an axial ratio of about 7–9. The ratio of the effective hydrodynamic volume to the partial specific volume is close to unity ( V e V = 1.1 ), suggesting that G-ADP actin in solution is not swollen. It is therefore proposed that the difference in the polymerizability and stability observed between G-ADP actin and G-ATP actin may be related to some subtle difference in the intramolecular structure of these monomeric proteins.

Fluorescence study of ϵ‐ADP bound to rabbit F‐actin: Structural change in the adenine subsite of F‐actin under the influence of heavy meromyosin

In a recent nanosecond pulse-fluorometric study of Factin+ADP [ 11, it was found that e-ADP is tightly bound in F-actin and the macromolecular motion of Factin is characterised by a correlation time as long as several microseconds. The study is extended in the present work so that the fluorescence of e-ADP bound to F-actin was measured in the presence of an Increasing amount of heavy meromyosin. Both static and time-dependent fluorescence measurements show that binding of heavy meromyosin to F-actin induces a cooperative structural change in the adenine subsite of F-actin. The cooperativity is enhanced by the combination of F-actin-e-ADP with tropomyosin.

Fluorescence and flow dichroism of F-actin-ϵ-ADP; the orientation of the ademine plane relative to the long axis of F-actin

The excitation polarization spectrum of epsilon-ADP bound to F-actin shows that two absorption dipoles at 260 nm and 340 nm are oriented in different directions relative to the emission dipole. On the other hand, the linear dichroism of F-actin-epsilon-ADP gives that the dichroic ratio of the bound epsilon-ADP is approximately constant (about-0.5) in the wavelength region form 250 to 350nm. Furthermore, the fluorescence polarization of epsilon-ADP bound to F-actin which is oriented in the field of flow shows that the emission dipole is nearly perpendicular to the long axis of F-actin. From these observations we conclude that the adenine plane of the bound nucleotide is almost perpendicular to the long axis of F-actin.

Nanosecond pulse fluorometry in polarized light of dansyl-L-cysteine linked to a unique SH group of F-actin; The influence of regulatory proteins and myosin moiety

The order of magnitude of the correlation time, which characterizes the dansyl cysteine residue linked to F-actin is ten times greater than the correlation time of the G-actin monomer [1]. Still it is much smaller than the correlation times of the F-actin polymer as a whole. The dansyl chromophore reveals that the C terminal end of the actin peptide chain, is mobile. As Ebashi and his co-workers have shown (13), Ca2+ triggers muscular contraction by acting on F-actin through the mediation of the regulatory proteins troponin and tropomyosin. By using spin label technique, Tonomura et al. [14] found that Ca2+ induces a conformational change on the troponin, tropomyosin actin complex. The quasi elastic scattering of laser light measurement of Fujime and Ishiwata [15] showed that troponin-tropomyosin F-actin has a rotational correlation time in the millisecond range which characterizes the flexibility of this complex; Ca2+ induces an increase of this flexibility. The present pulse fluorometry study shows an increase of mobility of the fluorescent probe induced by Ca2+. It seems difficult to correlate the results of the two kinds of measurements as long as we do not know the exact nature of the fluorescent kinetics unit.

Fluorescence study of N-(3-pyrene)maleimide conjugated to rabbit skeletal F-actin and plasmodium actin polymers.

A fluorescent probe N-(3-pyrene)maleimide was conjugated to rabbit skeletal F-actin at the site of most reactive sulfhydryl group (Cys-373). Its fluorescence anisotropy decay showed a single correlation time of 560 ns at 25 degrees C, which is in a very good agreement with the correlation time of the dansyl-L-cysteine group conjugated to the same site of F-actin reported very recently [Wahl, Ph., Mihashi, K, and Auchet, J-C. (1975) FEBS Lett. 8, 164-167]. Actin from plasmodia of myxomycates, Physarum polycepharum, was also conjugated with N-(3-pyrene) maleimide and the fluorescence anisotropy was compared with rabbit skeletal F-actin using the classical steady excitation method. It was found that the internal mobility of the magnesium polymer of plasmodium actin is remarkably larger than both plasmodium F-actin and rabbit skeletal F-actin.

Adiabatic compressibility of myosin subfragment-1 and heavy meromyosin with or without nucleotide

The partial specific adiabatic compressibilities of myosin subfragment-1 (S1) and heavy meromyosin (HMM) of skeletal muscle in solution were determined by measuring the density and the sound velocity of the solution. The partial specific volumes of S1 and HMM were 0.713 and 0.711 cm3/g, respectively. The partial specific adiabatic compressibilities of S1 and HMM were 4.2 x 10(-12) and 2.9 x 10(-12) cm2/dyn, respectively. These values are in the same range as the most of globular proteins so far studied. The result indicates that the flexibility of S1 region almost equals to that of HMM. After binding to ADP.orthovanadate, S1 and HMM became softer than their complexes with ADP. The bulk moduli of S1 and HMM were of the order of (4-6) x 10(10) dyn/cm2, which are very comparable with the bulk modulus of muscle fiber.