Troponin T fragments: physical properties and binding to troponin C.

Abstract

Fragments derived from rabbit skeletal troponin T (Tn-T) were tested for binding on a troponin C (Tn-C) – Sepharose affinity column in order to locate the binding site of Tn-C on Tn-T. The COOH-terminal fragments P2 (residues 159–209) and B2 (residues 206–258) were found to bind most strongly, confirming the earlier proposal (Pearlstone, J. R., Carpenter, M. R., Johnson, P. &Smillie, L. B. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1902–1906) that the highly basic COOH-terminal region of Tn-T may serve as a site of interaction for the acidic Tn-C protein. Results from circular dichroism experiments on the large fragments B1 (residues 1–205), B2, P1 (residues 91–154), and P2 indicated that most of the α-helical structure resides in CB2 (residues 71–151), the tropomyosin (Tm) binding site of Tn-T, while the remainder of the molecule including the Tn-C binding region (approximately residues 159–259) contains very little in the way of α and β structure. These results are in agreement with the secondary structural studies made previously (Pearlstone, J. R. &Smillie, L. B. (1977) Can. J. Biochem. 55, 1032–1038). The presence of calcium resulted in a stronger interaction between these fragments and Tn-C, illustrating the calcium-sensitive nature of this system. The addition of magnesium ions to the buffer system did not affect this binding.