Electron microscopic recording of myosin head power stroke in hydrated myosin filaments

Abstract

Muscle contraction results from cyclic attachment and detachment between myosin heads and actin filaments, coupled with ATP hydrolysis. Despite extensive studies, however, the amplitude of myosin head power stroke still remains to be a mystery. Using the gas environmental chamber, we have succeeded in recording the power stroke of position-marked myosin heads in hydrated mixture of actin and myosin filaments in a nearly isometric condition, in which myosin heads do not produce gross myofilament sliding, but only stretch adjacent elastic structures. On application of ATP, individual myosin heads move by ~3.3 nm at the distal region, and by ~2.5 nm at the proximal region of myosin head catalytic domain. After exhaustion of applied ATP, individual myosin heads return towards their initial position. At low ionic strength, the amplitude of myosin head power stroke increases to >4 nm at both distal and proximal regions of myosin heads catalytic domain, being consistent with the report that the force generated by individual myosin heads in muscle fibers is enhanced at low ionic strength. The advantages of the present study over other in vitro motility assay systems, using myosin heads detached from myosin filaments, are discussed.More than 60 years have passed since the monumental discovery that muscle contraction results from relative sliding between actin and myosin filaments, coupled with ATP hydrolysis 1,2 . It is now widely accepted that the myofilament sliding is caused by cyclic attachment-detachment between the myosin heads extending from myosin filaments and the sites on actin filaments 3 . Concerning performance of the myosin heads in producing the myofilament sliding, it is generally believed that a myosin head first attaches to actin, change their structure, i.e. power stroke, to produce myofilament sliding, and then detaches from actin A most effective approach to the structural changes of a myosin head may be to determine the unitary distance of myofilament sliding caused by a myosin head power stroke. In the contraction model of A.F. Huxley and Simmons 6 , each myosin head is assumed to have elastic element, while the myofilaments are rigid; when a muscle fiber contracts isometrically, its isometric tension results from the tension in stretched elastic element, which resides in individual myosin heads. The amount of extension of myosin head elastic element is therefore equal to the amplitude of myosin head power stroke (or step size) in the isometric condition, and can be estimated from the amount of quick decrease in fiber length to drop the

Enhancement of Force Generated by Individual Myosin Heads in Skinned Rabbit Psoas Muscle Fibers at Low Ionic Strength

“…Since the junctional peptide is located in between the two main actin binding sites of myosin heads [1], it seems clear that the antibody attached to the junctional peptide completely covers the two actin binding sites, so that formation of rigor linkages between actin and myosin head is no longer possible. If this explanation is correct, the result of our EC experiments, that individual myosin heads can perform ATP-induced power stroke [23], [24] may be taken to imply that, in the actin-myosin interaction cycle taking place in muscle, myosin heads do not pass through rigor configurations, which is generally believed to exist in the actomyosin ATPase reaction steps [26]. This idea is supported by our recent experiments that this antibody has no effect on both ATP-dependent vitro actin-myosin sliding and Ca 2+ -activated muscle fiber contraction [27].…”

“…In our EC experiments [23], individual myosin heads are position-marked by a monoclonal antibody directed to the junctional peptide between 50- and 20-kDa segments of myosin heavy chain [25]. Since the junctional peptide is located in between the two main actin binding sites of myosin heads [1], it seems clear that the antibody attached to the junctional peptide completely covers the two actin binding sites, so that formation of rigor linkages between actin and myosin head is no longer possible.…”

“…When only a small fraction of myosin heads are activated with ATP, the resulting myosin head movement should takes place in a nearly isometric condition; namely, each myosin head performes its power stroke by stretching adjacent sarcomeric strautures, as the overall filament sliding is suppressed by dominant actin-myosin rigor linkages. As a result, the amplitude of myosin head movement, recorded by the EC, was small, being about 3 nm [23]. Moreover, we have recently found that the amplitude of ATP-induced power stroke in individual myosin heasds increases to 4–5 nm, when the KCl concentration of experimental solution is reduced from 125 to <20 mM [24].…”

Evidence for the Essential Role of Myosin Subfragment-2 in Muscle Contraction: Functional Communication between Myosin Head and Subfragment-2

“…To position-mark individual myosin heads, colloidal gold particles (diameter, 15 nm; coated with protein A) were attached to myosin heads via site-directed monoclonal antibody to distal region of myosin head. Further details of the methods have been described elsewhere [8,9]. Figure 8 shows electron micrograph of myosinmanner.…”

“…Nevertheless, Harrington intended to demonstrate the important role of S-2 hinge region in muscle contraction, and prepared a polyclonal antibody to S-2 hinge region (anti-S-2 antibody), and showed that the antibody reduced Ca 2+ - Figure 6: Schematic diagram illustrating the helix to (random) coil transition cycle occurring in myosin-S-2 hinge region proposed by Harrington [9]. Helical and random coil structures of the S-2 hinge region are expressed as straight and zig-zag shapes, respectively [7].…”