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 Ca(2+)-binding domains relative to each other [Dobrowolski, Z., Xu, G.-Q., & Hitchcock-DeGregori, S.E. (1991) J. Biol. Chem. 266, 5703-5710]. Previous work showed that all mutants except dSEEE are partially defective in one part of the Ca2+ switch or the other. All mutants undergo Ca(2+)-dependent conformational changes as detected by changes in electrophoretic mobility, alpha-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 alpha-helix. There are differences among the mutants that do not correlate with function. All troponin C mutants show Ca(2+)-dependent interaction with troponin I and T in polyacrylamide gels. Troponin I-troponin C interaction was also analyzed by Ca(2+)-dependent increase in the monomer/excimer ratio of tropinin 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.
Dobrowolski
Z
Z
Department of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway 08854.
Xu
G Q
GQ
Chen
W
W
Hitchcock-DeGregori
S E
SE
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009154:Mutation; D009218:Myosins / Q000037:antagonists & inhibitors; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014336:Troponin / Q000235:genetics* / Q000378:metabolism; D019209:Troponin C; D019210:Troponin I
D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D009218:Myosins; D002118:Calcium