Nucleotide-induced flexibility change in neck linkers of dimeric kinesin as detected by distance Measurements using spin-labeling EPR.

Using dipolar continuous-wave and pulsed electron paramagnetic resonance methods, we have determined the distribution of the distances between two spin labels placed on the middle of each of the neck linkers of dimeric kinesin. In the absence of microtubules, the distance was centered at 3.3 nm, but displayed a broad distribution with a width of 2.

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.

Multicolor luciferase assay system: one-step monitoring of multiple gene expressions with a single substrate.

Reporter assays that use luciferase are widely employed for monitoring cellular events associated with gene expression. In general, firefly luciferase and Renilla luciferase are used for monitoring single gene expression. However, the expression of more than one gene cannot be monitored simultaneously by this system because one of the two reporting luciferases must be used as an internal control.

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.

ESR reveals the mobility of the neck linker in dimeric kinesin.

Conventional kinesin is a highly processive motor that converts the chemical energy of ATP hydrolysis into the unidirectional motility along microtubules. The processivity is thought to depend on the coordination between ATPase cycles of two motor domains and their neck linkers. Here we have used site-directed spin labeling electron spin resonance (SDSL-ESR) to determine the conformation of the neck linker in kinesin dimer in the presence and absence of microtubules.