Calbindin-D28K is a biologically essential calcium-binding protein of unknown tertiary structure that is required for normal neural function. The protein also plays an important role in the transport of calcium in epithelial cells of the intestine and kidney. The objective of this grant application is to determine the solution tertiary structure of calbindin-D28K in calcium-free and calcium-bound forms by use of high resolution nuclear magnetic resonance (NMR) spectroscopy, to determine what structural changes occur upon the binding of increasing amounts of calcium to the protein, and to thereby gain insights into the mechanism of action of the protein. The hypothesis of this grant application is that calbindin-D28K, a biologically essential, EF-hand, calcium-binding protein, has a unique tertiary structure which undergoes conformational changes upon binding calcium.
The specific aims are: (1) To obtain, using high-resolution nuclear magnetic resonance (NMR) spectroscopy, the solution tertiary structure of the calcium-free and the calcium-saturated forms of calbindin-D28K. (2) To determine the sequential structural changes which occur upon binding of calcium to calbindin-D28K. (3) To determine the effects of deletions of specific EF-hands in calbindin-D28K on the structure and calcium-binding properties of calbindin-D28K. The techniques to be used include: high resolution 1H, 15N and 13C nuclear magnetic resonance spectroscopy of the full-length and mutant calbindins; electrospray ionization mass spectrometry; lanthanide fluorescence spectroscopy; and intrinsic protein fluorescence determinations in the near and far UV-range. The project is of significance because the tertiary structure of calbindin-D28k in the presence and absence of calcium is unknown, the effects of initial calcium binding on global protein structure remain to be determined, and information on how the protein folds could provide insight into how it acts within cells. These findings will potentially be of significance in the understanding of neurodegenerative disorders, cerebellar function and long-term hippocampal potentiation.
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