Site-specific photo-cross-linking studies on interactions between troponin and tropomyosin and between subunits of troponin.

We have used the sulfhydryl-specific heterobifunctional photo-cross-linker 4-maleimidobenzophenone (BP-Mal) to study the interactions of rabbit skeletal tropomyosin with troponin and of the troponin subunits with each other. We found that alpha,alpha-tropomyosin specifically labeled at Cys-190 with BP-Mal photo-cross-links with all three subunits of troponin with decreasing cross-linking yields in the order of troponin T, troponin I, and troponin C. There was no apparent Ca2+ dependence in the cross-linking yields. In separate experiments, we found that troponin C labeled specifically at Cys-98 with BP-Mal photo-cross-links to both troponin I and troponin T in the two binary complexes, as well as in the ternary complex. Again, no Ca2+-dependent changes in the cross-linking yields were detectable. These results are in general agreement with the picture that troponin I and troponin T are in close contact with troponin C near its Cys-98 and that all three troponin subunits are in the proximity of Cys-190 of tropomyosin.

The conformation of the C-terminal region of actin: a site-specific photocrosslinking study using benzophenone-4-maleimide.

The site-specific photocrosslinker, benzophenone-4-maleimide, was used to label G-actin specifically at Cys-374, the penultimate residue from the C terminus. The resultant BP-G-actin was polymerized to form BP-F-actin, and both forms of actin were irradiated to activate the benzophenone moiety. We found that for BP-F-actin both intersubunit and intrasubunit photocrosslinks were formed. For BP-G-actin only a small amount of an internally photocrosslinked species was formed. These findings suggest that in the F-actin polymer, the C-terminal peptide is localized in a region between neighboring subunits. In contrast, in the G-actin monomer, the C-terminal peptide is relatively distant from the surface of the molecule.