Myosin isoforms show unique conformations in the actin-bound state (original) (raw)
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Strong Binding of Myosin Heads Stretches and Twists the Actin Helix
Biophysical Journal, 2005
Calculation of the size of the power stroke of the myosin motor in contracting muscle requires knowledge of the compliance of the myofilaments. Current estimates of actin compliance vary significantly introducing uncertainty in the mechanical parameters of the motor. Using x-ray diffraction on small bundles of permeabilized fibers from rabbit muscle we show that strong binding of myosin heads changes directly the actin helix. The spacing of the 2.73-nm meridional x-ray reflection increased by 0.22% when relaxed fibers were put into low-tension rigor (,10 kN/m 2 ) demonstrating that strongly bound myosin heads elongate the actin filaments even in the absence of external tension. The pitch of the 5.9-nm actin layer line increased by ;0.62% and that of the 5.1-nm layer line decreased by ;0.26%, suggesting that the elongation is accompanied by a decrease in its helical angle (;166°) by ;0.8°. This effect explains the difference between actin compliance revealed from mechanical experiments with single fibers and from x-ray diffraction on whole muscles. Our measurement of actin compliance obtained by applying tension to fibers in rigor is consistent with the results of mechanical measurements.
Insights into Actin-Myosin Interactions within Muscle from 3-D Electron Microscopy
2019
Much has been learned about the interaction between myosin and actin through biochemistry, in vitro motility assays and cryo-electron microscopy of F-actin decorated with myosin heads. Comparatively less is known about actin-myosin interactions within the filament lattice of muscle, where myosin heads function as independent force generators and thus most measurements report an average signal from multiple biochemical and mechanical states. All of the 3-D imaging by electron microscopy that has revealed the interplay of the regular array of actin subunits and myosin heads within the filament lattice has been accomplished using the flight muscle of the large waterbug Lethocerus sp. Lethocerus flight muscle possesses a particularly favorable filament arrangement that enables all the myosin cross-bridges contacting the actin filament to be visualized in a thin section. This review covers the history of this effort and the progress toward visualizing the complex set of conformational ch...
Journal of Muscle Research and Cell Motility, 1988
The structures of vertebrate skeletal muscles (particularly from frog and fish) in the rigor state are analysed in terms of the concept of target areas on actin filaments. Assuming that 100% of the heads are to be attached to actin in rigor, then satisfactory qualitative low-resolution modelling of observed X-ray diffraction data is obtained if the outer ends of these myosin heads can move axially (total range about 200.~) and azimuthally (total range less than 60 ~ from their original lattice sites on the myosin filament surface to attach in defined target areas on the actin filaments. On this basis, each actin target area comprises about four actin monomers along one of the two long-pitched helical strands of the actin filament (about 200 ~) or an azimuthal range of actin binding sites of about 100 ~ around the thin filament axis. If myosin heads simply label in a non-specific way the nearest actin monomers to them, as could occur with non-specific transient attachment in a 'weak binding' state, then the predicted X-ray diffraction pattern would comprise layer lines af the same axial spacings (orders of 429 A) as those seen in patterns from resting muscle.
Mechanics of actomyosin bonds in different nucleotide states are tuned to muscle contraction
2006
Muscle contraction and many other cell movements are driven by cyclic interactions between actin filaments and the motor enzyme myosin. Conformational changes in the actin-myosin binding interface occur in concert with the binding of ATP, binding to actin, and loss of hydrolytic by-products, but the effects of these conformational changes on the strength of the actomyosin bond are unknown. The force-dependent kinetics of the actomyosin bond may be particularly important at high loads, where myosin may detach from actin before achieving its full power stroke. Here we show that over a physiological range of rapidly applied loads, actomyosin behaves as a ''catch'' bond, characterized by increasing lifetimes with increasing loads up to a maximum at Ϸ6 pN. Surprisingly, we found that the myosin-ADP bond is possessed of longer lifetimes under load than rigor bonds, although the load at which bond lifetime is maximal remains unchanged. We also found that actomyosin bond lifetime is ultimately dependent not only on load, but loading history as well. These data suggest a complex relationship between the rate of actomyosin dissociation and muscle force and shortening velocity. The 6-pN load for maximum bond lifetime is near the force generated by a single myosin molecule during isometric contraction. This raises the possibility that all catch bonds between load-bearing molecules are ''mechanokinetically'' tuned to their physiological environment. catch bonds ͉ dynamic force spectroscopy ͉ laser trap ͉ myosin T he crystal structure of the myosin head (S1) (1) reveals how small-scale conformational changes within the hydrolytic site are converted into relatively large-scale changes producing movement. A globular ''motor domain'' occupies the bulk of the structure and contains both the nucleotide-and actin-binding sites (see Fig. 1). Of particular note is an ␣-helical extension of the heavy chain, or ''neck,'' protruding from the globular motor domain that acts as a rigid ''lever arm'' to amplify small movements arising in the nucleotide-binding pocket (2-5). Concomitant with phosphate release, rotation of the neck causes a step of an Ϸ5.5-nm ''working stroke'' and an isometric force of 0.7-9 pN (3, 6-10) that has been the subject of numerous single-molecule mechanics studies. Another feature of particular note within the motor domain is a cleft that divides the actin-binding site (2). This cleft is thought to close upon binding to actin (1, 2, 11-13), bringing into position residues on both sides of the cleft that are involved in strong binding to actin: the so-called R-site and A-site. Evidence suggests that there may be additional conformational changes in the actin-binding interface that accompany ADP release. The cleft at the actin-binding interface of smooth muscle myosin may close further upon ADP release (14, 15) and is accompanied by an increase in R-site flexibility (14, 16). Thus, although both the R-site and A-site are tightly bound to actin in the presence of ADP, upon ADP release from smooth muscle myosin the R-site adopts a more flexible conformation whereas the A-site remains tightly bound. EM reconstructions reveal no similar closure of the cleft in skeletal muscle myosin upon ADP release, although the resolution of the technique was limited. It does appear, however, that there are increases in R-site flexibility in skeletal muscle myosin upon ADP release (14).
Molecular mechanism of actin-myosin motor in muscle
Biochemistry (Moscow), 2011
The interaction of actin and myosin powers striated and smooth muscles and some other types of cell motility. Due to its highly ordered structure, skeletal muscle is a very convenient object for studying the general mechanism of the actin-myosin molecular motor. The history of investigation of the actin-myosin motor is briefly described. Modern con cepts and data obtained with different techniques including protein crystallography, electron microscopy, biochemistry, and protein engineering are reviewed. Particular attention is given to X ray diffraction studies of intact muscles and single mus cle fibers with permeabilized membrane as they give insight into structural changes that underlie force generation and work production by the motor. Time resolved low angle X ray diffraction on contracting muscle fibers using modern synchrotron radiation sources is used to follow movement of myosin heads with unique time and spatial resolution under near physio logical conditions.
Small segmental rearrangements in the myosin head can explain force generation in muscle
Biophysical Journal, 1996
Poisson-Boltzmann calculations of the distribution of electrostatic potentials around an actin filament in physiological-strength solutions show that negative isopotential surfaces protrude into the solvent. Each protrusion follows the actin two-start helix and is located on the sites implicated in the formation of the actomyosin complex. Molecular dynamic calculations on the Si portion of the myosin molecule indicate that in the presence of ATP the crystallographically invisible loops (comprising residues 624-649 and 564-579) remain on the surface, whereas in the absence of ATP they can move toward the actin-binding sites and experience electrostatic forces that range from 1 to 10 pN. The molecular dynamics calculations also suggest that during the ATP cycle there exist at least three states of electrostatic interactions between the loops and actin. Every time a new interaction is formed, the strain in the myosin head increases and the energy of the complex decreases by 2kT to 5kT. This can explain muscular contraction in terms of a Huxley-Simmons-type mechanism, while requiring only rearrangements of small mobile Si segments rather than the large shape changes in the myosin molecule postulated by the conventional tilting head model.
European Journal of Biochemistry, 1990
It was previously shown that tryptic digestion of subfragment 1 (Sl) of skeletal muscle myosins at 0°C results in cleavage of the heavy chain at a specific site located 5 kDa from the NH,-terminus. This cleavage is enhanced by nucleotides and suppressed by actin and does not occur at 25"C, except in the presence of nucleotide. Here we show a similar temperature sensitivity and protection by actin of an analogous chymotryptic cleavage site in the heavy chain of gizzard S1. The results support the view that the myosin head, in general, can exist in two different conformational states even in the absence of nucleotides and actin, and indicate that the heavy chain region 5 kDa from the NH,-terminus is involved in the communication between the sites of nucleotide and actin binding.
Muscle force is generated by myosin heads stereospecifically attached to actin
Nature, 1997
Muscle force is generated by myosin crossbridges interacting with actin. As estimated from stiffness and equatorial X-ray diffraction of muscle and muscle fibres, most myosin crossbridges are attached to actin during isometric contraction, but a much smaller fraction is bound stereospecifically. To determine the fraction of crossbridges contributing to tension and the structural changes that attached crossbridges undergo when generating force, we monitored the X-ray diffraction pattern during temperature-induced tension rise in fully activated permeabilized frog muscle fibres. Temperature jumps from 5-6 degrees C to 16-19 degrees C initiated a 1.7-fold increase in tension without significantly changing fibre stiffness or the intensities of the (1,1) equatorial and (14.5 nm)(-1) meridional X-ray reflections. However, tension rise was accompanied by a 20% decrease in the intensity of the (1,0) equatorial reflection and an increase in the intensity of the first actin layer line by appr...