Low angle X-ray diffraction: seeing cross-bridges move in reciprocal space
A powerful method for studying the movement of functioning muscle cross-bridges is to use an X-ray beam. When an X-ray beam travels through a muscle fibre, some of the photons are scattered by interaction with the proteins in muscle. As we have seen, the muscle cross-bridges have a repeating structure inside muscle fibres. The scattered photons interact in constructive and destructive ways resulting in a diffraction pattern that is recorded using an electronic detector. When the proteins in the fibre move, their movement is reflected in the diffraction pattern. The beauty of the diffraction pattern is that it records information with a spatial resolution of a few nanometres for X-rays with a wavelength of 0.1 nm. By using powerful X-ray sources such as that available at synchrotrons such as the European Synchrotron Radiation Facility (ESRF) in Grenoble, and fast detectors, a time resolution of about 0.1 ms can be reached. The disadvantages of this approach are that (i) the quality of the diffraction patterns is limited (the fibre structure is regular, but not as regular as in crystals, and the muscle mass is small resulting in relatively few diffracted photons), and (ii) a diffraction pattern is not the same as a real image of the muscle fibre, but is a map in Fourier space that needs to be converted into real space through modelling. This process requires assumptions and interpretation. An example diffraction pattern from mammalian muscle fibres is shown below. Each vertical line is a characterisitc 'layer-line' resulting from the helical packing of the filaments. M-labelled lines arise from the thick myosin filaments. The A-lines arise from the thin actin filaments. The darker vertical line in the centre is caused by a metal mask that absorbs some of the photons to avoid over-exposure of the much brighter 'equator'. The horizontal centre of the pattern, known as the meridian, shows bright spots related to the regular spacing of myosin crowns emanating from the thick filaments. The position of the spots and lines give absolute measurements of the repeating distances in the fibres, with a precision depending on the size and sharpness of the features. The quadrants represent fibres in different states: relaxed, rigor and contracting. The graphs on the right are integrations of x-ray intensites as explained in the figure legend.
(A) X-ray diffraction patterns obtained from three segments of a single muscle fiber in the relaxed state (upper left quadrant), in rigor (upper right quadrant), and during active contraction at ~30°C (both lower quadrants). The intensity of each pattern was scaled for fiber exposure in each state (300 ms, 700 ms, and 1200 ms, for relaxed, rigor, and active states, respectively) and symmetrically averaged; higher intensity is white, lower intensity is black. The equator is vertical and the meridian is horizontal. A vertical metal strip in front of the detector attenuates the equatorial reflections ~10-fold and prevents saturation of the detector; the inner part of the patterns at the meridional spacing of <0.087 nm−1 was attenuated by a factor of 3 to visualize strong and weak reflections on one diagram; meridional and layer line reflections mentioned in the text are labeled. (B–D) Meridional profiles of the off- meridional intensity for the same three diffraction patterns as in A in the regions of radial integration of 0.025–0.043 nm−1 (1,0 row line), 0.043–0.061 nm−1 (1,1 and 2,0 row lines), and 0.061–0.1 nm−1 (2,1, 3,0, 2,2, and 3,1 row lines), respectively; background subtracted. Gray dotted, black, and gray solid lines correspond to relaxed, rigor, and active states, respectively; the position of some layer lines is labeled in B. Koubassova NA, Bershitsky SY, Ferenczi MA, Tsaturyan AK. Biophys J. 2008 15; 95(6): 2880–2894.
When cross-bridges bind to the thin filaments in a regular orientation, the intensity of the first actin layer line (A1) increases. This is due to the increased protein mass associated with the thin filaments, and is clearly seen in the transition from relaxed to rigor states. Reciprocally, the first myosin layer line (M1) decreases in intensity. The measurements of changes in the intensity of the layer lines during contraction and length perturbations led to the development of a model of contraction in which initial binding of myosin cross-bridges to actin is 'non-stereospecific'. Cross-bridges have a high degree of azimuthal mobility, but contribute to the measured stiffness of the fibre. This non-stereospecific binding is rapidly followed by stereospecific binding: cross-bridges are locked onto the thin filaments; they contribute to enhanced A1 intensity and bear force. Lever arm movement is converted to additional force or sliding of the filaments (Ferenczi et al., 2005). This interpretation of the X-ray diffraction data suggests a dynamic role for the actin-myosin surface. More work is required to explore the binding modes of myosin to actin.
