Cadmium-substituted skeletal troponin C. Cadmium-113 NMR spectroscopy and metal binding investigations

P. D. Ellis, P. Strang, J. D. Potter

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Abstract

The binding of cadmium to skeletal troponin C (STnC) has been measured by equilibrium binding an by 113Cd NMR spectroscopy. The equilibrium binding experiments have shown that there are two cadmium binding sites on StnC with a high affinity for Cd2+ (K(Cd) ≃ 107 M-1) and two with a lower affinity for Cd2+ (K(Cd) ≃ 103 M-1). The former binding constant is comparable to Ca2+ binding to the Ca2+-Mg2+ (structural) sites of StnC and the latter is about a factor of one hundred less than Ca2+ binding to the Ca2+-specific (regulatory) sites of STnC. In the presence of Mg2+ the affinity of Cd2+ for the higher affinity sites was lowered, yielding a K(Mg) of approximately 103 M-1. These data clearly suggest that the two sites with high affinity for Cd2+ are the same as the Ca2+-Mg2+ sites (Zot, H., and Potter, J.D. (1982) J. Biol. Chem. 257, 7678-7683). The 113Cd NMR is shown to be temperature-dependent. The room temperature spectrum consists of two resonance at -107.8 and -112.7 ppm with respect to a 0.1 M solution of Cd(ClO4)2. Lowering the temperature to 4°C alters the cadmium exchange dynamics, and results in a four line 113Cd spectrum. The two new resonances at -103.1 and -109.8 ppm probably arise from cadmium binding to the Ca2+-specific (regulatory) sites on StnC; whereas, the resonances at -107.8 and -112.7 ppm correspond to cadmium binding at the Ca2+-Mg2+ (structural) sites, respectively. When the 113Cd2+-substituted protein was titrated with Ca2+, the two resonances corresponding to the high affinity sites were reduced in intensity, followed by a reduction in intensity of the lower affinity Cd2+ sites. Based on the assignments made here and the known binding constants of StnC for Ca2+ (Potter, J.D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628-4633) and the Cd2+ affinities reported here, one would not predict these results. Ca2+ shoud have first bound to the sites with the lower affinity Cd2+. Since the direct binding experiments clearly demonstrate that the high affinity CD2+ sites are the Ca2+-Mg2+ sites we can only conclude that Cd2+ binding to the protein (probably to the lower affinity Ca2+-specific sites) dramatically alters the affinity of the Ca2+-Mg2+ sites for Ca2+. It is suggested that an allosteric coupling network exists between all classes of binding sites.

Original languageEnglish
Pages (from-to)10348-10356
Number of pages9
JournalJournal of Biological Chemistry
Volume259
Issue number16
StatePublished - Jan 1 1984
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry

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