[NMR paper] Cardiac troponin I induced conformational changes in cardiac troponin C as monitored

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Cardiac troponin I induced conformational changes in cardiac troponin C as monitored by NMR using site-directed spin and isotope labeling.

Related Articles Cardiac troponin I induced conformational changes in cardiac troponin C as monitored by NMR using site-directed spin and isotope labeling.

Biochemistry. 1995 Oct 17;34(41):13343-52

Authors: Kleerekoper Q, Howarth JW, Guo X, Solaro RJ, Rosevear PR

Conformational changes in both free cardiac troponin C (cTnC) and in complex with a recombinant troponin I protein [cTnI(33-211), cTnI(33-80), or cTnI (86-211)] were observed by means of a combination of selective carbon-13 and spin labeling. The paramagnetic effect from the nitroxide spin label, MTSSL, attached to cTnC(C35S) at Cys 84 allowed measurement of the relative distances to the 13C-methyl groups of the 10 methionines of cTnC in the monomer or complex. All 10 1H-13C correlations in the heteronuclear single- and multiple-quantum coherence (HSMQC) spectrum of [13C-methyl] Met cTnC in the complex with cTnI(33-211) were previously assigned [Krudy, G. A., Kleerekoper, Q., Guo, X., Howarth, J. W., Solaro, R. J., & Rosevear, P. R. (1994) J. Biol. Chem. 269, 23731-23735]. In the presence of oxidized spin label, nine of the 10 Met methyl 1H-13C correlations of cTnC were significantly broadened in the cTnC(C35S) monomer. This suggests flexibility within the central helix, or interdomain D/E helical linker, bringing the N- and C-terminal domains in closer proximity than predicted from the crystallographic structure of TnC. In the spin-labeled cTnC(C35S). cTnI(33-211) complex only N-terminal Met methyl 1H-13C correlations of cTnC(C35S) were paramagnetically broadened beyond detection, whereas correlations for Met residues (103, 120, 137, and 157) in the C-terminal domain were not. Thus, complex formation with cTnI decreases interdomain flexibility and maintains cTnC in an extended conformation. This agrees with the recently published study suggesting that sTnC is extended when bound to sTnI [Olah, G. A., & Trewhella, J. (1994) Biochemistry 33, 12800-12806]. The recombinant N-terminal domain of cTnI, cTnI(33-80), gave similar results as observed with cTnI(33-211) when complexed with spin-labeled cTnC(C35S). However, complex formation with the C-terminal fragment, cTnI(86-211), which contains the inhibitory sequence, is insufficient to maintain cTnC extended to the amount observed with either cTnI(33-211) or cTnI(33-80); although compared to that observed in free cTnC, it does cause decreased flexibility in the interdomain linker. In the absence of the N-terminal domain of cTnI, there is a decrease in flexibility within the N-terminal domain of cTnC. Interestingly, the N-terminal domain of cTnC in the reduced spin-labeled complex with cTnI(86-211), in the presence of ascorbate, showed two distinct conformations which were not seen in the complex with cTnI(33-211).(ABSTRACT TRUNCATED AT 400 WORDS)

PMID: 7577919 [PubMed - indexed for MEDLINE]



Source: PubMed