Conformational changes in the metal-binding sites of cardiac troponin C induced by calcium binding

• Published in: Biochemistry (Easton) 1992-02, Vol.31 (6), p.1595-1602
• Main Authors: Krudy, George A, Brito, Rui M. M, Putkey, John A, Rosevear, Paul R
• Format: Article
• Language: English
• Subjects: 550201 - Biochemistry- Tracer Techniques; AMIDES; Amino Acid Sequence; AMINO ACIDS; Analytical, structural and metabolic biochemistry; BASIC BIOLOGICAL SCIENCES; binding; Binding Sites; Biological and medical sciences; BODY; calcium; Calcium - metabolism; CARBOXYLIC ACIDS; cardiac muscle; CARDIOVASCULAR SYSTEM; CHEMICAL SHIFT; CONFORMATIONAL CHANGES; Contractile proteins; Crystallization; effects on; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; GLYCINE; HEART; Holoproteins; Hydrogen Bonding; ISOTOPES; KETONES; LIGHT NUCLEI; Magnetic Resonance Spectroscopy; metals; Metals - metabolism; Molecular Sequence Data; Molecular Structure; Mutagenesis; Myocardium - chemistry; NITROGEN 15; NITROGEN ISOTOPES; NUCLEI; ODD-EVEN NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; ORGANS; OVERHAUSER EFFECT; Protein Conformation; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; STABLE ISOTOPES; TROPONES; Troponin - chemistry; Troponin - metabolism; Troponin C
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00121a003

Isotope labeling of recombinant normal cardiac troponin C (cTnC3) with 15N-enriched amino acids and multidimensional NMR were used to assign the downfield-shifted amide protons of Gly residues at position 6 in Ca(2+)-binding loops II, III, and IV, as well as tightly hydrogen-bonded amides within the short antiparallel beta-sheets between pairs of Ca(2+)-binding loops. The amide protons of Gly70, Gly110, and Gly146 were found to be shifted significantly downfield from the remaining amide proton resonances in Ca(2+)-saturated cTnC3. No downfield-shifted Gly resonance was observed from the naturally inactive site I. Comparison of downfield-shifted amide protons in the Ca(2+)-saturated forms of cTnC3 and CBM-IIA, a mutant having Asp65 replaced by Ala, demonstrated that Gly70 is hydrogen bonded to the carboxylate side chain of Asp65. Thus, the hydrogen bond between Gly and Asp in positions 6 and 1, respectively, of the Ca(2+)-binding loop appears crucial for maintaining the integrity of the helix-loop-helix Ca(2+)-binding sites. In the apo- form of cTnC3, only Gly70 was found to be shifted significantly downfield with respect to the remaining amide proton resonances. Thus, even in the absence of Ca2+ at binding site II, the amide proton of Gly70 is strongly hydrogen bonded to the side-chain carboxylate of Asp65. The amide protons of Ile112 and Ile148 in the C-terminal domain and Ile36 in the N-terminal domain data-sheets exhibit chemical shifts consistent with hydrogen-bond formation between the pair of Ca(2+)-binding loops in each domain of Ca(2+)-saturated cTnC3.