Optical properties of troponin, tropomyosin, and relaxing protein of rabbit skeletal muscle.

Optical rotatory dispersion (ORD), circular dichroism (CD), ultraviolet absorption, the fluorescence of troponin, tropomyosin, and relaxing protein were used as probes to detect structural changes resulting from the binding of troponin with tropomyosin to form a complex called relaxing protein. In the wave length region from 250 to 190 nm, ORD and CD spectra show that all the apparent band positions of the three proteins studied are similar to those reported for helical polypeptides. The helical contents estimated from ORD and CD troughs are 42, 97, and 72% for troponin, tropomyosin, and relaxing protein, respectively. In the ultraviolet region of the ORD and CD spectra, no change was observed in the secondary structure upon complexing troponin and tropomyosin. In addition, a stoichiometry of 1:l by weight is indicated since a mixture of troponin and tropomyosin yielded a spectrum which superimposes on that observed for relaxing protein. On the other hand, the CD spectra in the aromatic region (300 to 250 nm) indicate that the aromatic chromophores of either troponin or tropomyosin are perturbed. From an over-all change in the band intensities and from the disappearance of the positive 295 mn band upon binding of troponin to tropomyosin, it is suggested that the environment of the aromatic chromophores has been significantly modified. A perturbation associated with the binding of troponin to tropomyosin was also detected in the absorption spectrum. No change in the ionization constants for the phenolic groups of the tyrosine residues was observed upon formation of the relaxing protein. On the other hand, a shift of the fluorescence emission maximum of relaxing protein during storage suggests that tryptophan residues are perturbed in relaxing protein. It is, therefore, concluded that the perturbation of the aromatic residues results from a change in tryptophan rather than in the tyrosine residue environment.