Rotational dynamics of spin-labeled F-actin in the sub-millisecond time range

Thomas, David D.; Seidel, John C.; Gergely, J.

doi:10.1016/0022-2836(79)90259-6

Cited by 139 publications

(128 citation statements)

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“…The fluctuations, bending and twisting motions, are modulated by myosin and actin-binding proteins (13,14). The tighter binding of myosin to actin reduces the torsional motion of a small section of F-actin, as reported by standard transfer-EPR measurements (13,15). The change of the orientation of spin labels on F-actin during interaction with heavy meromyosin was also reported (10,16).…”

mentioning

confidence: 92%

“…Egelman et al (11) and later the workgroup in DeRosier's laboratory (12) emphasized the existence of variations in the twist along the axes of isolated filaments, which increases the fluctuations of approximately 10°in the azimuthal angle between adjacent monomers. The fluctuations, bending and twisting motions, are modulated by myosin and actin-binding proteins (13,14). The tighter binding of myosin to actin reduces the torsional motion of a small section of F-actin, as reported by standard transfer-EPR measurements (13,15).…”

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confidence: 99%

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The Flexibility of Actin Filaments as Revealed by Fluorescence Resonance Energy Transfer

Nyitrai¹,

Hild²,

Belágyi³

et al. 1999

Journal of Biological Chemistry

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The temperature profile of the fluorescence resonance energy transfer efficiency normalized by the fluorescence quantum yield of the donor in the presence of acceptor, f, was measured in a way allowing the independent investigation of (i) the strength of interaction between the adjacent protomers (intermonomer flexibility) and (ii) the flexibility of the protein matrix within actin protomers (intramonomer flexibility). In both cases the relative increase as a function of temperature in f is larger in calcium-F-actin than in magnesium-Factin in the range of 5-40°C, which indicates that both the intramonomer and the intermonomer flexibility of the actin filaments are larger in calcium-F-actin than those in magnesium-F-actin. The intermonomer flexibility was proved to be larger than the intramonomer one in both the calcium-F-actin and the magnesium-F-actin. The distance between Gln 41 and Cys 374 residues was found to be cation-independent and did not change during polymerization at 21°C. The steady-state fluorescence anisotropy data of fluorophores attached to the Gln 41 or Cys 374 residues suggest that the microenvironments around these regions are more rigid in the magnesiumloaded actin filament than in the calcium-loaded form.

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confidence: 92%

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confidence: 99%

The Flexibility of Actin Filaments as Revealed by Fluorescence Resonance Energy Transfer

Nyitrai¹,

Hild²,

Belágyi³

et al. 1999

Journal of Biological Chemistry

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“…Assays. Protein concentrations were determined as described (15). The fraction of S1 bound to actin was measured by sedimenting 200 gl of 1 ,uM S1 and various concentrations of actin , in the presence of 5 Mg2+*ATP under low or physiological ionic strength conditions (as defined above) in a Beckman TL-100 centrifuge for 8 min at 386,000 x g at 250C.…”

Section: Methodsmentioning

confidence: 99%

“…Chymotryptic S1 was prepared as described previously (14), except that the chymotryptic digestion time was 10 min. F-actin was prepared as previously described (15). S1 was spin-labeled with 4-maleimido-2,2,6,6,-tetramethyl-1-piperinyloxy (MSL; Aldrich) to the extent of 0.98 ± 0.02 label bound per head, with a specificity of 1.00 ± 0.04 SH1 groups blocked per bound label, as previously described (10).…”

Section: Methodsmentioning

confidence: 99%

Photolysis of a photolabile precursor of ATP (caged ATP) induces microsecond ratational motions of myosin heads bound to actin.

Berger

Svensson

Thomas

1989

Proc. Natl. Acad. Sci. U.S.A.

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To test the proposal that ATPase activity is coupled to the rotation of muscle cross-bridges (myosin heads attached to actin), we have used saturation-transfer EPR to detect the rotational motion of spin-labeled myosin heads (subfragment 1; S1) bound to actin following the photolysis of caged ATP (a photoactivatable analog of ATP). In order to ensure that most of the heads were bound to actin in the presence of ATP, solutions contained high (200 ,um) actin concentrations and were of low (36 mM) ionic strength. Sedimentation measurements indicated that 52 ± 2% of the spin-labeled heads were attached in the steady state of ATP hydrolysis during EPR measurements. Five millimolar caged ATP was added to the actin-Sl solution in an EPR cell in the dark, with no effect on the intense saturation-transfer EPR signal, implying a rigid actin-S1 complex. A laser pulse produced 1 mM ATP, which decreased the signal rapidly to a brief steady-state level that indicated only slightly less rotational mobility than that of free heads. After correcting for the fraction of free heads, we conclude that the bound heads have an effective rotational correlation time of 1.0 ± 0.3 ,Ps, which is about 100 times shorter (faster) than that in the absence of ATP. To our knowledge, this is the first direct evidence that myosin heads undergo rotational motion when bound to actin during the ATPase cycle. It is likely that similar cross-bridge rotations occur during muscle contraction.Although the sliding filament theory of muscle contraction was proposed in 1954 (1, 2), the exact nature of the actinmyosin interactions that cause the filaments to slide past one another remains unclear. H. E. Huxley suggested in 1969 (3) that the most probable source of force generation is the rotation of the myosin head when bound to actin, and in 1971 A. F. Huxley and Simmons (4) proposed that the myosin head undergoes submillisecond rotations while bound to actin during isometric contraction. X-ray diffraction experiments on active muscle suggest that myosin heads remain in close proximity to actin during contraction (5), but there has been no direct evidence for the rotation of myosin heads bound to actin during the ATPase cycle.Previous work on spin-labeled myosin heads in isometrically contracting muscle fibers, using conventional EPR to measure orientation (6) and saturation-transfer EPR (ST-EPR) to measure microsecond rotational motions (7), has shown that 80% of the heads are orientationally disordered and mobile on the microsecond time scale. One interpretation of these results is that the mobile heads are detached from the actin filament, but an alternative explanation is that they are bound to actin in a rotationally dynamic state. It is difficult to distinguish between these two possibilities in complex cycling systems as muscle fibers or myofibrils (8), since the fraction of attached heads is ambiguous. Although the stiffness of fibers in contraction is greater than 50% of the rigor value, whether labeled or not, stiffness is not necessarily ...

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“…However, this assumption is not necessarily correct, because the actin filament is a double-helical polymer of globular actin monomers (13,14). Spectroscopic (12,(15)(16)(17) and electron microscopic studies (18) have suggested that the elastic property of an actin filament is largely anisotropic in the directions of twisting, bending, and stretching, i.e., the torsional rigidity is much smaller than the bending and longitudinal ones, assuming the actin filament to be a homogeneous rod, whereas the normal mode analysis based on the atomic structure of actin has shown much larger torsional rigidity than those suggested by these studies (19).Here, we have directly determined the torsional rigidity of actin filaments and the actin-actin bond breaking force under torsion by manipulating single actin filaments with optical tweezers and microneedles, respectively. Developments in video-assist fluorescence microscopy have enabled direct observation of single actin filaments labeled with fluorescent phalloidin in solution (20).…”

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confidence: 99%

Torsional rigidity of single actin filaments and actin–actin bond breaking force under torsion measured directly by in vitro micromanipulation

Tsuda

Yasutake²,

Ishijima³

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

264

204

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Knowledge of the elastic properties of actin filaments is crucial for considering its role in muscle contraction, cellular motile events, and formation of cell shape. The stiffness of actin filaments in the directions of stretching and bending has been determined. In this study, we have directly determined the torsional rigidity and breaking force of single actin filaments by measuring the rotational Brownian motion and tensile strength using optical tweezers and microneedles, respectively. Rotational angular f luctuations of filaments supplied the torsional rigidity as (8.0 ؎ 1.2) ؋ 10 ؊26 Nm 2 . This value is similar to that deduced from the longitudinal rigidity, assuming the actin filament to be a homogeneous rod. The breaking force of the actin-actin bond was measured while twisting a filament through various angles using microneedles. The breaking force decreased greatly under twist, e.g., from 600-320 pN when filaments were turned through 90؇, independent of the rotational direction. Our results indicate that an actin filament exhibits comparable f lexibility in the rotational and longitudinal directions, but breaks more easily under torsional load.Actin is a major protein involved in a variety of cellular motile events and in the maintenance of cell shape and form. Determining its elastic properties in the polymeric state is central to an understanding of its function (1, 2). Recently, actin filaments have been measured to be several-fold more flexible longitudinally in vitro (3) and in muscle (4-6) than many models of muscle contraction have assumed (7). This finding would seem to require reconsideration of aspects of such models (8-10). Because of its helical structure, an actin filament should experience not only longitudinal but also torsional loads during interactions with myosin (11). To explain the mechanism of force generation, it is also important to know the elastic behavior of actin filaments during torsion. The torsional rigidity of actin filament can be estimated based on its bending rigidity, assuming the actin filament to be a homogenous rod (12). However, this assumption is not necessarily correct, because the actin filament is a double-helical polymer of globular actin monomers (13,14). Spectroscopic (12,(15)(16)(17) and electron microscopic studies (18) have suggested that the elastic property of an actin filament is largely anisotropic in the directions of twisting, bending, and stretching, i.e., the torsional rigidity is much smaller than the bending and longitudinal ones, assuming the actin filament to be a homogeneous rod, whereas the normal mode analysis based on the atomic structure of actin has shown much larger torsional rigidity than those suggested by these studies (19).Here, we have directly determined the torsional rigidity of actin filaments and the actin-actin bond breaking force under torsion by manipulating single actin filaments with optical tweezers and microneedles, respectively. Developments in video-assist fluorescence microscopy have enabled direct observat...

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Rotational dynamics of spin-labeled F-actin in the sub-millisecond time range

Cited by 139 publications

References 48 publications

The Flexibility of Actin Filaments as Revealed by Fluorescence Resonance Energy Transfer

The Flexibility of Actin Filaments as Revealed by Fluorescence Resonance Energy Transfer

Photolysis of a photolabile precursor of ATP (caged ATP) induces microsecond ratational motions of myosin heads bound to actin.

Torsional rigidity of single actin filaments and actin–actin bond breaking force under torsion measured directly by in vitro micromanipulation

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