Structural responses to the photolytic release of ATP in frog muscle fibres, observed by time‐resolved X‐ray diffraction

Abstract

Photolytic release of ATP from an inactive precursor has provided an approach to studying some of the elementary steps of energy transduction in muscle (Goldman et al. 1982). Photolytic release of nucleotides is an effective way to initiate contraction in cellular systems in which the internal structural organisation plays an essential role in defining function. In muscle cells, the three-dimensional packing of arrays of protein filaments is integral to in vivo muscular function. The order also enables time-resolved information about protein movements to be obtained by means of lowangle X-ray diffraction (Huxley et al. 1983).Here we show, with a time resolution of 1 ms, the structural changes in permeabilised fibres isolated from frog skeletal muscle in response to rapid addition of ATP resulting from DMB-caged ATP photolysis (Thirlwell et al. 1994), in the absence and presence of Ca¥, at 5-6°C. The purpose of the study is to determine the changes in structure, at high time resolution, which accompany force generation when a muscle fibre in the rigor state is activated by photogeneration of ATP. These changes characterise the nature of molecular changes which are responsible for the generation of force and for shortening. Journal of Physiology (1999), 520.3, pp. 681-696 681 Structural responses to the photolytic release of ATP in frog muscle fibres, observed by time-resolved X-ray diffraction 1. Structural changes following the photolytic release of ATP were observed in single, permeabilised fibres of frog skeletal muscle at 5-6°C, using time-resolved, low-angle X-ray diffraction. The structural order in the fibres and their isometric function were preserved by cross-linking 10-20% of the myosin cross-bridges to the thin filaments. 2. The time courses of the change in force, stiffness and in intensity of the main equatorial reflections (1,0) and (1,1), of the third myosin layer line (M3) at a reciprocal spacing of (14·5 nm)¢ on the meridian and of the first myosin-actin layer line (LL1) were measured with 1 ms time resolution. 3. In the absence of Ca¥, photolytic release of ATP in muscle fibres initially in the rigor state caused the force and stiffness to decrease monotonically towards their values in relaxed muscle fibres. 4. In the presence of Ca¥, photolytic release of ATP resulted in an initial rapid decrease in force, followed by a slower increase to the isometric plateau. Muscle fibre stiffness decreased rapidly to •65% of its value in rigor. 5. In the absence of Ca¥, changes on the equator, in LL1 and in M3 occurred with a time scale comparable to that of the changes in tension and stiffness. 6. In the presence of Ca¥, the changes on the equator and LL1 occurred simultaneously with the early phase of tension decrease. The changes in the intensity of M3 (IM3) occurred on the time scale of the subsequent increase in force. The time courses of the changes in tension and IM3 were similar following the photolytic release of 0·33 or 1·1 mÒ ATP. However the gradual return towards the rigor state began earlie...

Insight into the actin-myosin motor from x-ray diffraction on muscle

“…A short dip in I M3 followed by its rise was resolved using fast DMB-caged ATP. It was also found that the rise in I M3 has the same rate constant as tension rise (12). These observations indicate that force generation is accompanied by a rearrangement of the cross-bridge orientation.…”

“…Flash photolysis of caged-ATP in the presence of Ca 2+ did not reveal a delay between ATP-induced dissociation of myosin heads from actin and their reattachment as estimated by the intensity of the 1st actin layer line A1 (12,14). This probably results from insufficient spatial and temporal resolution of those experiments that makes it difficult to distinguish between the M1 and A1 layer lines.…”

“…No significant difference in the rate of ATP-induced acto-myosin dissociation between one-headed S1s and two-headed myosin molecules in muscle was found (ibid.). Fast DMB-caged ATP provides faster structural responses of muscle fibers than conventional NPE-caged ATP (12). Experiments using arrays of single muscle fibers and a very bright light source showed that the intensity of the beating actin-myosin layer line AM +1 at ~10.4 nm decays slower than those of all actin layer lines upon ATP liberation in the absence of Ca 2+ (15).…”

“…Early x-ray experiments with flash photolysis of caged-ATP were focused on relaxation kinetics of the equatorial intensities (10,(133)(134)(135). Later with the use of more advanced radiation sources and 2D x-ray detectors the intensities of the M3 meridional reflection and some layer lines were also resolved in the absence and in the presence of Ca 2+ (11,136,137,(12)(13)(14)(15). It was found that the decay of the rigor structure of actin filaments decorated by strongly bound myosin heads has a time course similar to that found in solution if the rate of ATP liberation and binding of caged-ATP to myosin are taken into account (15).…”

“…An attenuator strip was placed along the equator in front of a CCD detector to avoid its saturation; experimental methods were described earlier (9). Synchronization of structural changes in a large population of myosin molecules in a muscle specimen can be achieved by fast perturbations of muscle length (4,5), load (6), temperature (7,8,9), concentrations of ATP (10)(11)(12)(13)(14)(15) or Ca 2+ (16). These experimental approaches together with x-ray interferometry (3) and addition of exogenous proteins into muscle fibers (17)(18)(19)(20) which had their cell membrane treated to make them permeable revealed new structural features of the actin-myosin molecular motor and of its regulation.…”