Proton NMR study of rabbit skeletal muscle troponin C: magnesium-induced conformational change

Abstract

Binding of Mg2+ to rabbit skeletal muscle troponin C (TnC) is studied by means of two-dimensional (2D) 1H NMR spectroscopy. Using the sequence-specific resonance assignment method we assign several resonances of TnC in the Mg2(+)-saturated state. Assigned resonances are used as probes of the following titration experiments: (1) Mg2+ titration of apo-TnC, (2) Mg2+ titration of Ca2TnC, and (3) Mg2+ titration of Ca4TnC. In experiment 1, the slow-exchange behavior is observed for resonances of Phe99, Asp107, Gly108, Tyr109, Ile110, Asp111, His125, Gly144, Arg145, Ile146, Asp147, and Phe148 located at the high-affinity Ca2(+)-binding sites in the C-terminal-half domain. In experiments 1 and 2, the fast-exchange behavior is observed for resonances of Gly32, Asp33, Ser35, Gly68, Thr69, and Asp71 located at the low-affinity Ca2(+)-binding sites in the N-terminal-half domain. These results suggest that Mg2+ ions bind to the N domain as well as the C domain. In experiment 3, no spectral change is observed for all above-mentioned residues in the C domain and also for Gly32 and Gly68 in the N domain. It can be concluded that all Ca2(+)-binding sites in both the N and C domains can bind Mg2+ ions. No significant change is observed for resonances of Phe23, Ile34, Val68, and Phe72 in experiments 1 and 2. These results suggest that Mg2+ binding to the N domain does not induce conformational change in the hydrophobic region of the N domain. 2D-NMR spectra and Mg2(+)-titration data suggest that the antiparallel beta-sheet conformation is formed in both the N and C domains when Mg2+ ions bind to the two domains.

A 1H NMR study of a ternary peptide complex that mimics the interaction between troponin C and troponin I

“…The partial assignment of whole turkey skeletal s-TnC was accomplished using standard methods employing one-dimensional 'H NMR calcium titration data and 2D DQF-COSY and NOESY data and will not be presented. Partial proton assignments of the C-terminal domain of rabbit s-TnC and whole rabbit s-TnC have been published (Drabikowski et al, 1985;Tsuda et al, 1988Tsuda et al, , 1990 and are in agreement with those assignments made of turkey s-TnC. These proteins are highly homologous in sequence and share a similarity in sequence and structure to CaM.…”

Ca(2+)-dependence of structural changes in troponin-C in demembranated fibers of rabbit psoas muscle

“…For example, through the interaction between the two domains of TnC, the Mg2+-sensitivity of the second transition of the fluorescence-pCa relations determined with highly stretched fibers might stem from the Mg2+-sensitivity of the first transition, hence from the Mg2+-sensitivity of the high-affinity sites. This possibility gains strength from the results of NMR spectroscopy performed during the titration with Ca2+ of TnC and its proteolytic fragments (Drakenberg et al, 1987; for differing view, see Tsuda et al, 1990). A thermodynamic evaluation of this possibility awaits the determination of the free energy coupling for the binding of Ca2+ to the two domains of TnC when in the regulatory complex.…”

Microtiter plate monoclonal antibody epitope analysis of Ca2+- and Mg2+-induced conformational changes in troponin C

“…This observation implicated that Mg 2+ was able to weakly bind the N-domain low affinity sites of TnC in the absence of Ca 2+ . A previous two-dimensional 1 H NMR study [25] investigated the binding of Mg 2+ to intact rabbit fast TnC. In the presence of 2 or 4 bound Ca, fast-exchange behavior was observed for residues located at the low-affinity Ca 2+ -binding sites in the N-domain.…”