THE STRUCTURE OF CALBINDIN-D28K

Project: Research project

Project Details

Description

DESCRIPTION (Adapted from the Applicant's Abstract): 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.
StatusFinished
Effective start/end date2/15/011/31/07

ASJC

  • Medicine(all)