Time-resolved diffraction studies of muscle using synchrotron radiation

Abstract

Muscle contraction is one of those biological phenomena that we can all appreciate in our everyday lives. Sometimes it is when we are resting quietly and are aware of our heartbeat. At other times it may be when we are exerting ourselves and become short of breath, or when we exercise for a long period and our muscles start to ache.The way in which muscles produce force has exercised the minds of philosophers and scientists at least since the days of Erasistratus in the third century BC. Nowadays, of course, we know a very great deal about muscle structure, physiology and biochemistry, but we still do not know exactly what the molecular process is that produces movement. An ideal way of probing this process would be to be able to obtain signals from the relevant molecules as they actually go through their normal force-generating routine in an active muscle. The spatial dimensions involved are in the region of 1-50 nm, thus precluding the use of light microscopy, and the time regime is microseconds to milliseconds. Techniques with the appropriate spatial resolution might be electron microscopy and x-ray diffraction, but electron microscopy cannot yet be carried out on living tissue. X-ray diffraction methods can clearly have the right sort of spatial resolution, but what about recording diffraction patterns in the very short times involved (say 1 ms)? It is here that the high flux from synchrotron storage rings comes into its own. Using synchrotron radiation from, say, the SRS at the CCLRC Daresbury Laboratory it is possible to record x-ray diffraction patterns from living muscles in the millisecond time regime and to follow how these diffraction patterns change as the muscles go through typical contraction cycles. Unfortunately, x-ray diffraction is not a direct imaging method; the observed distribution of diffracted intensity needs to be interpreted in some way to give useful information on the spatial relationships of the force-generating molecules.This review details the practical methods involved in recording time-resolved x-ray diffraction patterns from active muscles and the theoretical approaches that are being used to interpret the diffraction patterns that are obtained. The ultimate aim is to produce a series of time-sliced images of the changing molecular arrangements and shapes in the muscle as force is produced; together these images will form 'Muscle-The Movie'.

Axial Dispositions and Conformations of Myosin Crossbridges Along Thick Filaments in Relaxed and Contracting States of Vertebrate Striated Muscles by X-ray Fiber Diffraction

“…In the present study, an idealized helical symmetry of the myosin molecules in the filaments was assumed, except in the perturbed region; in frog skeletal muscle, three successive crossbridges consisting of two myosin heads in pairs per crown are arrayed to form a three-stranded helix with a helical repeat of ∼129 nm with an axial rise of ∼14.3 nm and nine crown levels within the helical repeating unit. 10 In the perturbed region, it was assumed for simplicity that each level in the three successive crowns deviates only axially from the regular disposition, and this set of crowns was treated as a unit cell that is helically arranged. Therefore, each level of crossbridges rotates azimuthally by 40°when going to the next crown level in both regions.…”

“…[23][24][25][26] Very recently, perplexing intensity behaviors of the myosin-based meridional reflections upon the application of rapid length perturbation to contracting whole muscle have been reported. 27,28 As has been discussed, 7,10,[29][30][31] the difficulty in interpreting these data is that the intensities of myosin meridional reflections are determined by multiple factors, including interference between the two heads of an individual crossbridge, interference between the diffraction from the crossbridge projecting from the backbone and that from structures within the backbone itself, as well as the changing interference by the mirror image structure of the thick filaments across the M-line. We have no clear information on the relative importance of these various contributions that would allow unambiguous reconstruction of the axial mass projection of the myosin crossbridges onto the fiber axis.…”

The Myosin Filament Superlattice in the Flight Muscles of Flies: A-band Lattice Optimisation for Stretch-activation?

“…In summary, a typical IFM myosin filament would give intensity on the meridian at layer-lines l = ±16, ±32, ±48 and so on and off-meridional peaks on layer-lines l = ±6, ±10, ±26 and so on. For an explanation of helical diffraction see, for example, 18 Harford & Squire, 19 Squire 20 and Squire & Knupp. 21 The actin filaments in IFM, with 28 actin monomers in 13 turns of the genetic helix and a repeat of 2 × 38.7 nm, would be expected to give a strong meridional peak at the 28th order of 2 × 387 nm (2 × 38.7 nm/28 = 2.76 nm; this is the 84th order of 232 nm) and strong off-meridional layer-lines at the 13th order of 2 × 38.7 nm (=5.95 nm; 39th order of 232 nm) and the 15th order (27 -13) of 2 × 38.7 nm (=5.16 nm; 45th order of 232 nm).…”

Biomolecular Imaging at High Spatial and Temporal Resolution In Vitro and In Vivo

“…b A discontinuous helix can be described by adding z and φ, which define the translation and rotation between subunits. Redrawn from [70] [70] excellent reviews, see [71,72]). The start-number of the helix of each layer line can be calculated with the use of cylindrical Bessel functions, which describe the distance from the meridian of the first major peak in the FT.…”