Cross-bridge orientation in skeletal muscle measured by linear dichroism of an extrinsic chromophore

Linear dichroism of chromophoric labels attached to myosin heads has been used to establish cross-bridge orientation in myofibrils and muscle fibers. Generalized expressions were obtained for the dichroic ratio of a circularly symmetrical assembly of chromophores viewed through high apertures. The theoretical expressions were used to estimate the angle Θ of the absorption dipole of the dye relative to the myofibrillar axis. Myosin subfragment-1 has been labeled with tetramethyl rhodamine and diffused into the I-band of myofibrils; endogenous muscle myosin has been labeled directly. Dichroism has been measured from these preparations in the absence (rigor) and presence of MgATP and its analogs. In rigor, angle Θ was 80 °. Relaxed and contracted preparations displayed no dichroism, suggesting a high degree of cross-bridge disorder. MgAMP-PNP^† † Abbreviations used: MgAMP-PNP, Mg-5′-adenylylimidodiphosphate; AP₅A, P¹, P⁵-diadenosine-5′-pentaphosphate; IATR, iodoacetamidotetramethylrhodamine; IAF, 5-iodoacetamidofluorescein; 1,5-IAEDANS, 5-iodoacetamidoethylaminonaphthalene-1-sulfonic acid; HMM, heavy meromyosin; S-1, myosin subfragment-1; TRFAD, time-resolved fluorescence anisotropy decay; EGTA, ethyleneglycolbis(β-aminoethyl ether)N,N′-tetraacetic acid. and MgPP_i imposed on the cross-bridges a distribution intermediate between rigor and relaxation. In the presence of MgADP the preparations showed strong dichroism of the opposite direction to that present in rigor. No detachment of the cross-bridges occurred under these conditions and the effect was not due to the rotational displacement of the attached dye by the nucleotide. It is concluded that the formation of a ternary complex myosin-MgADP-actin makes it possible to detect a large local deformation imposed on the cross-bridge by nucleotide binding, which results in a change of the spatial attitude of the mobile region of the protein by about 40 °.