Back to Search
Start Over
The effects of N helix deletion and mutant F29W on the Ca2+ binding and functional properties of chicken skeletal muscle troponin.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 1994 May 27; Vol. 269 (21), pp. 14988-94. - Publication Year :
- 1994
-
Abstract
- To assess the structural and functional significance of the N helix (residues 3-13) of avian recombinant troponin C (rTnC), we have constructed NHdel, in which residues 1-11 have been deleted, both in rTnC and in the spectral probe mutant F29W (Pearlstone, J. R., Borgford, T., Chandra, M., Oikawa, K., Kay, C. M., Herzberg, O., Moult, J., Herklotz, A., Reinach, F. C., and Smillie, L. B. (1992) Biochemistry 31, 6545-6553). Comparison of the far- and near-UV CD spectra (+/- Ca2+) of F29W and F29W/NHdel and titration of the Ca(2+)-induced ellipticity and fluorescence changes indicates that the deletion has little effect on the global fold of the molecule but reduces the Ca2+ affinity of the N domain, but not the C domain, by 1.6-1.8-fold. Comparisons of the mutants NHdel, F29W, and F29W/NHdel with rTnC have been made using several functional assays. In reconstituted troponin-tropomyosin actomyosin subfragment 1 and myofibrillar ATPase systems, both F29W and NHdel have significantly reduced Ca(2+)-activated enzymatic activities. These effects are cumulative in the double mutant F29W/NHdel. On the other hand, maximal isometric tension development in Ca(2+)-activated reconstituted skinned fibers is not affected with F29W and NHdel, although the Ca2+ sensitivity of NHdel in this system is markedly reduced. We conclude that both mutations, NHdel and F29W, are functionally deleterious, possibly affecting interactions of the N domain with troponin I and/or T.
- Subjects :
- Animals
Base Sequence
Binding Sites
Chickens
Circular Dichroism
Molecular Sequence Data
Mutagenesis, Site-Directed
Oligodeoxyribonucleotides
Peptide Fragments chemistry
Peptide Fragments genetics
Peptide Fragments metabolism
Sequence Deletion
Spectrophotometry, Ultraviolet
Troponin chemistry
Troponin genetics
Troponin C
Calcium metabolism
Muscles metabolism
Troponin metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 269
- Issue :
- 21
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 8195134