ANALYSIS OF THE REGULATORY AND STRUCTURAL DEFECTS OF TROPONIN-C CENTRAL HELIX MUTANTS

Five deletion mutants of the D/E linker region of the troponin C central helix were tested for conformational and functional differences from wild-type troponin C. The mutants were in the region 87KEDAKGKSEEE97: dEDA, dKG, dKGK, dKEDAKGK, and dSEEE, designed to change the length of the central helix and the orientation of the Ca2+-binding domains relative to each other [Dobrowolski, Z., Xu, G.-Q., & Hitchcock-DeGregori, S. E. (I 99 1) J. Biol. Chem. 266, 5703-57 1 01. Previous work showed that all mutants except dSEEE are partially defective in one part of the Ca2+ switch or the other. All mutants undergo Ca2+-dependent conformational changes as detected by changes in electrophoretic mobility, a-helix content, and hydrophobic exposure. Deletions of the central helix do not extensively alter the thermal stability of troponin C, as determined by temperature-dependent loss of a-helix. There are differences among the mutants that do not correlate with function. All troponin C mutants show Ca+-dependent interaction with troponin I and T in polyacrylamide gels. Troponin 1-troponin C interaction was also analyzed by Ca2+-dependent increase in the monomer/excimer ratio of troponin I and relief of inhibition of the actomyosin S1 ATPase. While all mutants retain basic function, dKGK, dKEDAKGK, and dEDA have altered interaction with troponin I in the absence of Ca2+. dSEEE differs in conformation from wild type, but it is normal in functional assays. This conserved region of the D/E linker is not required for interaction with troponin I in the presence or absence of urea.