Spin label studies of tropomyosin

Article abstract of DOI:10.1021/bi00681a019

Studies are reported on nitroxide spin labeled tropomyosin. The labels attach to sulfhydryl groups and to amino groups. The amino spins are highly mobile, the sulfhydryl much less so. Spin count studies show an average of 0.5 labeled sulfhydryl/tropomyosin molecule and only 0.15 labeled amino group/molecule. The spectra ae used to study the denaturation of tropomyosin by guanidine hydrochloride. The information obtained reveals the course of denaturation at sites near the sulfhydryl group. It is found that these sites are more susceptible to guanidine than the bulk of the molecule; denaturation at the sulfhydryl sites is complete by 1.5 M guanidine, whereas optical studies indicate the molecule as a whole is not completely denatured until the concentration reaches 3.5 M. Spectra are also shown of tropomyosin fibers oriented variously with respect to the applied magnetic field. Strong orientation effects are seen and these indicate that the sulfhydryl attached spins (but not the amino attached spins) have a definite orientation in the fiber. Interpretation of the spectra reveals that the normal to the nitroxide plane is inclined to the fiber axis at an angle of 50°. Circular dichroism (CD) studies in the tyrosine region also reveal drastic changes wih guanidine denaturation, confirming the idea that denaturation produces pronounced increased in mobility at the β carbon (as in the sulfhydryl case). A strong negative band existing only in helical tropomyosin at pH's where the tyrosines are uncharged appears to be due to interaction of tyrosines with the helical backbone, whereas the appearance of a strong positive CD band at 250 nm at high pH (11) seems to be ascribable to interaction between the charged phenolic groups and the dissymmetric backbone α carbon atom.