Assignment and calcium dependence of methionyl epsilon C and epsilon H resonances in cardiac troponin C.
The 10 Met methyl groups in recombinant cardiac troponin (cTnC) were metabolically labeled with [13C-methyl]Met and detected as 10 individual cross-peaks using two-dimensional heteronuclear single- and multiple-quantum coherence (HSMQC) spectroscopy. The epsilon C and epsilon H chemical shifts for all 10 Met residues were sequence-specifically assigned using a combination of HSMQC and systematic conversion of the Met residues to Leu. The only negative functional consequence of these changes was seen when both Met 45 and 81 were mutated. Binding of Ca2+ to the high affinity C-terminal sites III and IV induced relatively large changes in the epsilon H and epsilon C chemical shifts of all Met residues in the C-terminal domain as well as small but significant changes in the chemical shifts of epsilon H Met 47 and Met 81 in the N-terminal half of cTnC. Binding of Ca2+ to the low affinity N-terminal site II induced large changes in the epsilon H and epsilon C chemical shifts of Met 45, Met 80, and Met 81. Binding of Ca2+ to site II had no effect on the chemical shifts of Met residues located in the C-terminal domain. The nature of the chemical shift changes of Met residues in the N- versus the C-terminal halves of cTnC were consistent with different Ca(2+)-induced conformational changes in these domains. Thus, the assigned methyl Met chemical shifts can serve as useful structural markers to study conformational transitional in free cTnC and potentially after association with small ligands, peptides, and other troponin subunits.
Lin
X
X
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77030.
Krudy
G A
GA
Howarth
J
J
Brito
R M
RM
Rosevear
P R
PR
Putkey
J A
JA
D002118:Calcium / Q000737:chemistry; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014336:Troponin / Q000737:chemistry*; D019209:Troponin C
D011994:Recombinant Proteins; D014336:Troponin; D019209:Troponin C; D008715:Methionine; D002118:Calcium