Single molecule fluorescence polarization studies of the myosin light chain domain

A detailed understanding of the molecular mechanism of force production has been elusive. A working hypothesis is that the motor domain (MD) of myosin remains rigidly bound to actin while the light chain domain (LCD) rotates about a hinge (Huxley and Kress, 1985; Cooke, 1986; Rayment et al, 1993; Goldman, 1998) in a nucleotide dependent manner. Recently, time-resolved fluorescence polarization (TRFP) (Hopkins et al, 1998; Corrie et al., 1999; Hopkins et al, 2002) was utilized to measure directly, the tilting of crossbridges in muscle fibers during active contraction. Signals reflecting tilting were surprisingly small (∼3°). Presumably, the smallness of the angle change detected is due to ensemble averaging. To address this issue, we initiated TRFP experiments at the single molecule level. Total internal reflection microscopy was combined with TRFP such that conformational changes could be measured on individual molecules in real time. Here we present data from decorated actin filaments similar to the “tracks” described by Toyoshima et al. (1989). Data were collected with 40 ms time resolution. Simultaneous detection of 3-dimensional orientation and extent of motion on the microsecond and nanosecond timescales are demonstrated. Direct evidence of two HMM rigor orientations was obtained. Comparison of MgS1 and HMM orientations suggests that the HMM heads are strained when they are both bound to actin