The microscopic and wavelength-dependent fluorescence polarization signals from the 5-[[[(iodoacetyl)amino]ethyl]amino]naphthalene-l-sulfonic acid (1,5-IAEDANS) labeled subfragment 1 of myosin (S-l) decorating muscle fibers in rigor and in the presence of MgADP are measured. Using microscopic fluorescence polarization, we select a small uniform volume (~0.1 μm3) from the muscle fiber and detect a high degree of angular order. From these data we show that the probe angular distribution from fibers in rigor is quantitatively different from that present when MgADP is bound to S-l. Using wavelength-dependent fluorescence polarization, we vary the wavelength of the excitation light and thereby change the direction that the probe absorption dipole makes with a reference frame fixed in S-l. From these data we show that the binding of MgADP to S-l causes an angular reorientation of S-l relative to the actin filament. The difference between the angular distribution of probes for the rigor vs. MgADP states cannot be accounted for by the addition of random probes. The microscopic fluorescence polarization experiments suggest that the earlier attempts to distinguish a rigor from a MgADP probe angular distribution by using the 1,5-IAEDANS probe failed due to the lower resolution of the optical technique employed. The wavelength-dependent fluorescence polarization experiments indicate that the probe dipole orientation on S-l, at the typically used excitation and emission wavelengths, is not ideal for detecting the orientation change in the rigor to MgADP angular transition.
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