title: The mechanism of Ca(2+)-coordination in the EF-hand of TnC, by cassette mutagenesis.
abstract: Genetic engineering of TnC and skinned fiber physiology on rabbit psoas muscle are combined to study the mechanisms of Ca(2+)-binding in the EF-hand in TnC. Of the six coordinating positions (X,Y,Z,-Y,-X & -Z) for Ca(2+)-binding in the loop, the X position is invariably occupied by an aspartate, and the -Z position by a glutamate. X-ray analysis has indicated that both oxygen atoms of the beta-carboxylate in aspartate (in X) are extensively hydrogen bonded to other residues in the loop. When this aspartate in site II was replaced by a glutamate (gamma-carboxylate), Ca(2+)-binding was annihilated, and the mutant was unable to regulate force development in the fiber. Similarly, glutamate for aspartate exchange in the -Z position of site I also inactivated the site as well as its function in skinned fiber. Mutations in the Y position indicated that a glutamate was unacceptable in place of aspartate but that an asparagine was acceptable. The Ca(2+)-sensitivity with asparagine was also similar to that of the wild type. The study indicates a powerful approach for defining the physicochemical principles governing Ca-coordination and sensitivity in Ca-binding proteins. Furthermore, by comparison with findings on chemically synthesized peptides, the results show that behavior of the EF-hand in TnC is modified by quaternary structure of the molecule.
authors.affiliation: Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461.
authors.forename: A
authors.initials: A
authors.lastname: Babu
authors.forename: H
authors.initials: H
authors.lastname: Su
authors.forename: J
authors.initials: J
authors.lastname: Gulati
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D009132:Muscles / Q000737:chemistry*; D016254:Mutagenesis, Insertional; D014336:Troponin / Q000737:chemistry / Q000235:genetics*; D019209:Troponin C
chemical_list: D014336:Troponin; D019209:Troponin C; D002118:Calcium