The mechanism of Ca(2+)-coordination in the EF-hand of TnC, by cassette mutagenesis.
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.
Babu
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A
Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461.
Su
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H
Gulati
J
J
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
D014336:Troponin; D019209:Troponin C; D002118:Calcium