Ca²⁺-induced conformational change and aggregation of chromogranin A

Chromogranin A, the most abundant protein in bovine adrenal chromaffin granules, bound calmodulin in a Ca²⁺-dependent manner, and the calmodulin-binding property was utilized to purify chromogranin A. Chromogranin A has been described in the past as a "random-coil polypeptide" with little α-helix or β-sheet conformation. However, circular dichroism measurements with pure, native chromogranin A revealed relatively high α-helical contents (40% at the intravesicular pH of 5.5). Fluorescence studies confirmed previous observations that chromogranin A binds Ca²⁺ with low affinity. Considering the high concentration of Ca²⁺ in the secretory vesicle, the effect of Ca²⁺ on the secondary structure and self-association of chromogranin A was examined. Ca²⁺ induced a decrease of α-helicity of chromogranin A from 40 to 30% at pH 5.5. In contrast, at pH 7.5 the same amount of Ca²⁺ increased α-helicity of the protein from 25 to 40%. Boiling of the adrenal extract, a commonly used purification procedure for chromogranin A, resulted in the isolation of conformationally distinct chromogranin A molecule. Unlike secretory protein-I of the parathyroid gland (Gorr, S.-V., Dean, W. L., Radley, T. L., and Cohn, D. V. (1988) Bone Mineral 4, 17-25), chromogranin A aggregated rapidly in the presence of Ca²⁺. The extent and rate of aggregation were highly dependent on Ca²⁺ concentration. Although both the rate and extent of aggregation at pH 7.5 were much lower than those at pH 5.5, aggregation of chromogranin A proceeded at both pH's. In this respect, chromogranin A differs from human chromogranin C which was shown by Gerdes et al. (Gerdes, H.-H., Rosa, P., Phillips, E., Baeuerle, P. A., Frank, R., Argos, P., and Huttner, W. B. (1989) J. Biol. Chem. 264, 12009-12015) to aggregate at pH 5.2 but not at pH 7.4.