The effects of N helix deletion and mutant F29W on the Ca2+ binding and functional properties of chicken skeletal muscle troponin.
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.
Chandra
M
M
Department of Biochemistry, University of Alberta, Edmonton, Canada.
da Silva
E F
EF
Sorenson
M M
MM
Ferro
J A
JA
Pearlstone
J R
JR
Nash
B E
BE
Borgford
T
T
Kay
C M
CM
Smillie
L B
LB
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D002942:Circular Dichroism; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013056:Spectrophotometry, Ultraviolet; D014336:Troponin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019209:Troponin C
D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D014336:Troponin; D019209:Troponin C; D002118:Calcium