Small-angle X-ray scattering (SAXS) can be used to study changes in protein conformation in the native environment of a living cell. The structural information obtained by SAXS is often complementary to that obtained by protein crystallography; SAXS has lower spatial resolution, but can reveal the dynamics of structural changes in relation to protein function.

Recent experiments carried out on ID2 (SAXS beamline) have shown that it is possible to measure angstrom-scale motion of a protein by SAXS. This study probed the motion of myosin heads during contraction of single intact muscle fibres. The axial X-ray diagram from a muscle fibre shows reflections that index on the approximate 43 nm quasi-helical repeat of the myosin filaments (M1, M2, M3; Figure 3). The prominent M3 reflection comes from the regular 14.5 nm repeat of the motor domain of myosin (the myosin head) along the myosin filaments. Owing to the high brilliance and unprecedented collimation of the X-ray beam on ID2, it is shown that this reflection, during contraction, is composed of two closely-spaced sub-peaks (Figure 3a).

These two sub-peaks are due to X-ray interference between the two arrays of myosins in each filament (Figure 4). The myosin filament (black) is bipolar; it is designed to pull neighbouring actin filaments (blue) towards its centre, and this relative sliding between the actin and myosin filaments is responsible for muscle contraction. The centres of the two arrays of myosin heads in each filament are 870 nm apart during active contraction. This interference distance, corresponding to 59.5 repeats of the 14.5 nm axial periodicity, is maintained with atomic precision by interactions between the elongated tails of the myosins in the filament backbone.

The interference distance changes when the myosin head binds to actin and drives filament sliding by tilting towards the centre of the myosin filament [1]. The interference effect gives a very precise measure of this motion, so that changes in the interference distance by about 1 Å can be measured from the relative intensities of the two sub-peaks of the M3 reflection. The method has already been used to measure the change in conformation of the myosin head between isometric contraction and the rigor state observed in the absence of ATP (Figures 3 and 4). By combining large area image plates and precise fast shuttering, this interference technique is now being used to characterise the sub-millisecond motion of the myosin heads that are responsible for force generation.

[1] I. Dobbie, M. Linari, G. Piazzesi, M. Reconditi, N. Koubassova, M.A. Ferenczi, V. Lomabrdi, M. Irving, Nature, 396, 383-387 (1998).

V. Lombardi (a), M. Irving (b), G. Piazzesi (a), M. Linari (a), M. Reconditi (a), M.E. Vannicelli Casoni (a), I. Dobbie (b), T. Narayanan (c) P. Boesecke (c).

(a) Università di Firenze (Italy)
(b) King's College London (UK)
(c) ESRF