Abstract
Defining the structural changes involved in the myosin cross-bridge cycle on actin in active muscle by X-ray diffraction will involve recording of the whole two dimensional (2D) X-ray diffraction pattern from active muscle in a time-resolved manner. Bony fish muscle is the most highly ordered vertebrate striated muscle to study. With partial sarcomere length (SL) control we show that changes in the fish muscle equatorial A-band (10) and (11) reflections, along with (10)/(11) intensity ratio and the tension, are much more rapid than without such control. Times to 50% change with SL control were 19.5 (±2.0) ms, 17.0 (±1.1) ms, 13.9 (±0.4) ms and 22.5 (±0.8) ms, respectively, compared to 25.0 (±3.4) ms, 20.5 (±2.6) ms, 15.4 (±0.6) ms and 33.8 (±0.6) ms without control. The (11) intensity and the (10)/(11) intensity ratio both still change ahead of tension, supporting the likelihood of the presence of a head population close to or on actin, but producing little or no force, in the early stages of the contractile cycle. Higher order equatorials (e.g., (30), (31), and (32)), more sensitive to crossbridge conformation and distribution, also change very rapidly and overshoot their tension plateau values by a factor of around two, well before the tension plateau has been reached, once again indicating an early low-force cross-bridge state in the contractile cycle. Modelling of these intensity changes suggests the presence of probably two different actin-attached myosin head structural states (mainly low-force attached and rigor-like). No more than two main attached structural states are necessary and sufficient to explain the observations. We find that 48% of the heads are off actin giving a resting diffraction pattern, 20% of heads are in the weak binding conformation and 32% of the heads are in the strong (rigor-like) state. The strong states account for 96% of the tension at the tetanus plateau.
Highlights
Despite tremendous progress in understanding the contractile mechanism in muscle and other myosin motors (Wulf et al [1]; Squire et al [2]), it is still uncertain exactly how many myosin headsBiology 2016, 5, 41; doi:10.3390/biology5040041 www.mdpi.com/journal/biologyBiology 2016, 5, 41 interact at the same time with actin filaments in muscle to utilise the energy associated with ATP hydrolysis and produce muscular contraction
What are the structural changes in the myosin heads that are associated with force production and movement? The myosin head has two major domains within it, a globular motor domain which is the enzymatic and actin attachment part of the head, and a long lever arm consisting of a long α–helix surrounded by two light chains
We have shown that the Plaice fin muscle is a viable preparation to use for recording time-resolved low-angle X-ray diffraction pattern from active whole muscle with sarcomere length control
Summary
Biology 2016, 5, 41 interact at the same time with actin filaments in muscle to utilise the energy associated with ATP hydrolysis and produce muscular contraction. It is not known how many different attached states there are and how much force is produced by each state. During some stage of this process force and movement (if the muscle is free to shorten) are produced Binding of another ATP molecule splits the heads from actin and a further cycle of hydrolysis and actin attachment can take place. Because of the weakness of the sampled myosin layer-lines, the 100 ms active frame was found to give the best background fit, with no detectable reflection intensity in the fitted background. For the second experimental session, the background, of the same form, increased in intensity slightly as the muscle contracted, so the background was scaled throughout the time series to give a better fit to the diffraction patterns
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