The interaction of troponin molecules on the thin filament with Ca(2+) plays a key role in regulating muscle contraction. To characterize the structural changes of troponin caused by Ca(2+) and crossbridge formation, we recorded the small-angle x-ray intensity and the myoplasmic free Ca(2+) concentration using fluo-3 AM in the same frog skeletal muscle during twitch elicited by a single electrical pulse at 16 degrees C. In an overstretched muscle, the intensity of the meridional reflection from troponin at 1/38.5 nm(-1) began to change at 4 ms after the stimulus, reached a peak at 10 ms, and returned to the resting level with a halftime of 25 ms. The concentration of troponin-bound Ca(2+) began to increase at 1-2 ms after the stimulus, reached a peak at 5 ms, and returned to the resting level with a halftime of 40 ms, indicating that troponin begins to change conformation only after a sizable amount of Ca(2+) has bound to it, and returns to the resting structure even when there is still some bound Ca(2+). In a muscle with a filament overlap, crossbridge formation appears to slow down Ca(2+) release from troponin and have a large effect on its conformation.
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