Structural changes during activation of frog muscle studied by time-resolved X-ray diffraction.

The pattern given by contracting frog muscle can be followed with high time resolution using synchrotron radiation as a high-intensity X-ray source. We have studied the behaviour of the second actin layer-line (axial spacing of approximately 179 A) at an off-meridional spacing of approximately 0.023 A-1, a region of the diagram that is sensitive to the position of tropomyosin in the thin filaments. In confirmation of earlier work, we find that there is a substantial increase in the intensity of this part of the pattern during contraction. We find that the reflection reaches half its final intensity about 17 milliseconds after the stimulus at 6 degrees C. The changes in the equatorial reflections, which arise from movement of crossbridges towards the thin filaments, occur with a delay of about 12 to 17 milliseconds relative to this change in the actin pattern. In over-stretched muscle, where thick and thin filaments no longer overlap, the changes in the actin second layer-line still take place upon stimulation with a time course and intensity similar to that observed at full overlap. This indicates that tropomyosin movement, in response to calcium binding to troponin, is the first structural step in muscular contraction, and is the prerequisite for myosin binding. A change in intensity similar to that found in contracting muscle is seen in rigor, where tropomyosin is probably locked in the active position. During relaxation the earlier stages in the decrease in intensity of the second actin layer-line take place significantly sooner after the last stimulus than tension decay. In over-stretched muscles the intensity decay is appreciably faster than in the same muscles at rest length, where attached crossbridges may interfere with the return of tropomyosin to its resting position.