Dynamic structures of motor proteins myosin and kinesin, and switch protein troponin as detected by SDSL-ESR.

We have studied biological nano-machines, motor and switch proteins operating as supramolecular complexes by electron spin resonance (ESR) and found key features of their molecular movements. In all the systems, the specific movements of elements or domains were detected and quite dynamic at nanometer scale. We have observed two broad but distinct orientations, separated by a 25 degrees axial rotation, of a spin label attached specifically to the light chain (LC) domain of myosin motor in the muscle fibers. The distribution became only narrower upon muscle activation. ESR spectrum from the spin label of the neck-linker of dimeric kinesin motor consisted of immobilized and mobilized components and did not exhibit nucleotide-dependent mobility change. The distance between two labels of kinesin dimer was also measured by spin dipole-dipole interaction, showing a broad distribution and a nucleotide-dependent change on the nanometer scale (>1.5 nm). These results suggest that two LC domains of myosin and two neck linkers of kinesin play a similar role for sliding movement using two conformations. The spin label of the skeletal (Tn)-I regulatory domain (TnIreg) showed a large mobility change by Ca2+ ion suggesting a Ca-induced switch movement of TnIreg. Spin dipole-dipole interaction showed that in reconstituted muscle fibers both skeletal and cardiac TnC undergo Ca2+-induced structural change that is thought to be essential for TnIreg movement. We also succeeded in fixing the newly-synthesized bifunctional spin label rigidly on the TnC molecule in solution, indicating that we can determine the precise coordinate of the spin principal axis of troponin on the oriented filament.

Orientation and motion of myosin light chain and troponin in reconstituted muscle fibers as detected by ESR with a new bifunctional spin label.

Using electron spin resonance, we have studied dynamic structures of myosin neck domain and troponin C by site-directed spin labeling. We observed two broad but distinct orientations of a spin label attached specifically to a single cysteine (cys156) on the regulatoy light chain (RLC) of myosin in relaxed skeletal muscle fibers. The two probe orientations, separated by a 25 degrees axial rotation, did not change upon muscle activation, but orientational distributions became narrower substantially, indicating that a fraction of myosin heads undergoes a disorder-to-order transition of the myosin light chain domain upon force generation and muscle contraction. These results provide insight into the mechanism how myosin heads move their domains to translocate an actin filament. Site-directed spin-labeling was achieved by cysteine residues of human cardiac troponin C (TnC). Spin dipole-dipole interaction showed that free TnC undergoes a global structural change (extended-to-compact) by Ca2+ or Mg2+. The spectra from the spin labels at N-terminal half domain were broad and almost identical in parallel and perpendicular orientations of fiber, suggesting that the N-terminal of TnC molecule is flexible or disoriented with respect to the filament axis. We also succeeded, for the first time, in fixing the newly-synthesized bifunctional spin label rigidly on TnC molecule in solution (either in +/- Ca2+), giving a promise that we can determine the precise coordinate of the spin principal axis on protein surface.