Muscle force is generated by myosin crossbridges interacting with actin. As estimated from stiffness and equatorial X-ray diffraction of muscle and muscle fibres, most myosin crossbridges are attached to actin during isometric contraction, but a much smaller fraction is bound stereospecifically. To determine the fraction of crossbridges contributing to tension and the structural changes that attached crossbridges undergo when generating force, we monitored the X-ray diffraction pattern during temperature-induced tension rise in fully activated permeabilized frog muscle fibres. Temperature jumps from 5-6 degrees C to 16-19 degrees C initiated a 1.7-fold increase in tension without significantly changing fibre stiffness or the intensities of the (1,1) equatorial and (14.5 nm)(-1) meridional X-ray reflections. However, tension rise was accompanied by a 20% decrease in the intensity of the (1,0) equatorial reflection and an increase in the intensity of the first actin layer line by approximately 13% of that in rigor. Our results show that muscle force is associated with a transition of the crossbridges from a state in which they are nonspecifically attached to actin to one in which stereospecifically bound myosin crossbridges label the actin helix.
Structural changes induced by Joule temperature jumps (T-jumps) in frog muscle fibers were monitored using time-resolved x-ray diffraction. Experiments made use of single, permeabilized fibers that were fully activated after slight cross-linking with 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide to preserve their structural order. After T-jumps from 5-6 to approximately 17 degrees C and then on to approximately 30 degrees C, tension increased by a factor of 1.51 and 1.84, respectively, whereas fiber stiffness did not change with temperature. The tension rise was accompanied by a decrease in the intensity of the (1, 0) equatorial x-ray reflection by 15 and 26% (at approximately 17 and approximately 30 degrees C) and by an increase in the intensity of the M3 myosin reflection by 20% and 41%, respectively. The intensity of the (1,1) equatorial reflection increased slightly. The peak of the intensity on the 6th actin layer line shifted toward the meridian with temperature. The intensity of the 1st actin layer line increased from 12% (of its rigor value) at 5-6 degrees C to 36% at approximately 30 degrees C, so that the fraction of the cross-bridges labeling the actin helix estimated from this intensity increased proportionally to tension from approximately 35% at 5-6 degrees C to approximately 60% at approximately 30 degrees C. This suggests that force is generated during a transition of nonstereo-specifically attached myosin cross-bridges to a stereo-specific binding state.
A set of programs has been developed for rapid collection of x-ray intensity data from protein and virus crystals with a commercially available two-dimensional focused geometry electronic detector. The detector is compact and portable, with unusually high spatial resolution comparable to that used in oscillation photography. It has allowed x-ray data collection on weakly diffracting crystals with large unit cells, as well as more conventional "diffractometer-quality" crystals. The quality of the data is compared with that from oscillation photography and automated diffractometry in the range of unit cells from 96.3 to 383.2 angstroms. Isomorphous and anomalous difference Pattersons, based on detector data, are shown for a variable surface glycoprotein mercury derivative and for a repressor-DNA bromine derivative, which has been solved at 7 angstroms with detector data only.
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...
Slow data acquisition rates have, in the past, limited the use of X-ray diffraction for characterization of polymeric materials. Photon counting techniques yield quantitative data in digital form for computer analysis. However, a great deal of data acquisition time is required when data is taken sequentially; i.e., when each intensity determination (at a particular goniometer setting) requires a separate time interval, during which intensity at other angle values is ignored. The problem is particularly acute for oriented polymers since two or more diffraction angles are involved: The Bragg angle along with at least one orientation angle. For this reason, an area-imaging (two-dimensional) proportional counter has been developed for use with a four-circle X-ray diffraction system. Although basically a single-crystal unit, this goniometer has been used in this laboratory and others for studies of oriented polymers. The receiving pinhole collimator and aperture have been removed, and the area-imaging counter has been mounted on the detector arm track. The original receiving aperture is still used for alignment, and the area detector Is positioned with its center at the receiving aperture center position.
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