LUMINESCENCE RESONANCE ENERGY-TRANSFER MEASUREMENTS IN MYOSIN

Myosin is thought to generate force by a rotation between the relative orientations of two domains. Direct measurements of distances between the domains could potentially confirm and quantify these conformation al changes, but efforts have been hampered by the large distances invo lved. Here we show that luminescence resonance energy transfer (LRET), which uses a luminescent lanthanide as the energy-transfer donor, is capable of measuring these long distances. Specifically, we measure di stances between the catalytic domain (Cys(707)) and regulatory light c hain domain (Cys(108)) of the myosin head. An energy transfer efficien cy of 21.2 +/- 1.9% is measured in the myosin complex without nucleoti de or actin, corresponding to a distance of 73 Angstrom, consistent wi th the crystal structure of Rayment et al. Upon binding to actin, the energy transfer efficiency decreases by 4.5 +/- 1.0%, indicating a con formational change in myosin that involves a relative rotation and/or translation of Cys(707) relative to the light chain domain. Addition o f ADP also alters the energy transfer efficiency, likely through a rot ation of the probe attached to Cys(707). These results demonstrate tha t LRET is capable of making accurate measurements on the relatively la rge actomyosin complex, and is capable of detecting conformational cha nges between the catalytic and light chain domains of myosin.