RESUMEN

An enantiomeric pair of C2-chiral bifunctionalised spin labels having a pyrrolidine nitroxide moiety, whose configurations were determined by X-ray crystal diffraction analysis, was prepared and applied to troponin C whose binding mode of double disulfide linkage was proved by EPR spectroscopy.

Asunto(s)

Carbono/química , Marcadores de Spin , Troponina C/química , Animales , Pollos , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Estructura Terciaria de Proteína , Estereoisomerismo , Troponina C/síntesis química

RESUMEN

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.

Asunto(s)

Espectroscopía de Resonancia por Spin del Electrón/métodos , Fibras Musculares Esqueléticas/metabolismo , Cadenas Ligeras de Miosina/química , Troponina/química , Actinas/química , Animales , Calcio/química , Cisteína/química , Cistina/química , Escherichia coli/metabolismo , Humanos , Magnesio/química , Músculo Esquelético/metabolismo , Músculos/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Miosinas/química , Distribución Normal , Estructura Terciaria de Proteína , Conejos , Marcadores de Spin , Troponina C/química