Bovine adrenal medulla troponin-C. Demonstration of a calcium-dependent conformational change.

A high affinity calcium-binding protein has recently been purified from the adrenal medulla (Kuo, I.C.Y., and Coffee, C.J. (1976) J. Biol. Chem. 251, 1603-1609). This protein is closely related in its chemical and physical properties to troponin-C (TN-C) of muscle tissue. Further examination of the adrenal medulla protein indicates that the removal of calcium is accompanied by a marked change in the conformation. This change in structure is similar, if not identical, to the calcium-dependent conformational change which has been described for skeletal muscle TN-C (Murray, A.C., and Kay, C.M. (1972) Biochemistry 11, 2622). The far ultraviolet circular dichroism spectrum of native adrenal medulla calcium-binding protein (AM-CBP) shows characteristic helical ellipticity bands at 222 and 207 nm. The helical content, as estimated from these data, is between 40 and 45%. Removal of calcium is accompanied by a change in ellipticity which corresponds to a decrease from 40 to 20% in the helical content. The near-ultraviolet circular dichroism spectrum shows negative dichroic bands at 262 and 268 nm which are characteristic of phenylalanine. These bands are relatively insensitive to changes in the calcium ion concentration. Sedimentation velocity studies likewise are indicative of a calcium-dependent structural alteration. The sedimentation coefficient of the native protein was observed to be 1.89 S. Similar measurements performed in the presence of 3 mM ethylene glycol bis(beta-aminoethyl ether) N,N, N', N'-tetraacetic acid (EGTA) gave a sedimentation coefficient of 1.50 S. The molecular weight, as determined by sedimentation equilibrium studies, was 16,000 regardless of whether the measurements were made in the presence of CaCl2 or EGTA. From the elution properties of AM-CBP on Sephadex G-100, the Stokes radius was observed to be 19.8 A in the presence of calcium and 21.9 A in the presence of EGTA. All of these changes which were induced by the addition of EGTA were completely reversible by the readdition of excess CaCl2. These data suggest that the removal of calcium from AM-CBP is accompanied by a pronounced conformational change which occurs without a molecular weight change. The decreased sedimentation coefficient, the increased Stokes radius, and the reduced helical content, which are observed for the apoprotein, indicate that removal of calcium results in a transformation from a compact symmetrical structure to one that is less ordered and more asymmetrical.