The Mechanism of Ca2+-Coordination in the EF-Hand of TnC, by Cassette Mutagenesis

  • Árvind Babu
  • Hong Su
  • Jagdish Gulati
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 332)


Genetic engineering of TnC and skinned fiber physiology on rabbit psoas muscle are combined to study the mechanisms of Ca2+-binding in the EF-hand in TnC. Of the six coordinating positions (X,Y,Z,-Y,-X & -Z) for Ca2+-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 β-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 (γ-carboxylate), Ca2+-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 Ca2+-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.


Quaternary Structure Regulatory Light Chain Force Regulation Extensive Hydrogen Bonding Skinned Fiber 
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Copyright information

© Springer Science+Business Media New York 1993

Authors and Affiliations

  • Árvind Babu
    • 1
    • 2
  • Hong Su
    • 1
    • 2
  • Jagdish Gulati
    • 1
    • 2
  1. 1.The Molecular Physiology LaboratoryDivision of CardiologyBronxUSA
  2. 2. Departments of Medicine & Physiology/BiophysicsAlbert Einstein College of MedicineBronxUSA

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