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Papers In Press, published online ahead of print May 28, 2002
Department of Biochemistry, Vanderbilt University, Nashville, TN 37232-0146
Corresponding Author: walter.chazin{at}vanderbilt.edu
Caltractin (centrin) is a member of the calmodulin subfamily of EF-hand Ca2+-binding proteins that is an essential component of microtubule organizing centers in many organisms ranging from yeast and algae to humans. The protein contains two homologous EF-hand Ca2+-binding domains linked by a flexible tether; each domain is capable of binding two Ca2+ ions. In an effort to search for domain-specific functional properties of caltractin, the two isolated domains were sub-cloned and expressed in E. coli. Ca2+ binding affinities and the Ca2+-dependence of biophysical properties of the isolated domains were monitored by UV, CD, and NMR spectroscopies. Comparisons to the corresponding results for the intact protein showed that the two domains function independently of each other in the absence of any target proteins. Ca2+-dependent binding of a peptide fragment from the yeast Kar1p protein to the isolated domains and intact caltractin shows that the two domains interact with the peptide independently, with the C-terminal domain binding more strongly than the N-terminal domain. Measurements of the macroscopic Ca2+ binding constants show that only the N-terminal domain has sufficient apparent Ca2+ affinity in vitro (1-10 mM) to be classified as a traditional calcium sensor in signal transduction pathways. However, investigation of the microscopic Ca2+ binding events in the C-terminal domain by NMR spectroscopy revealed that the observed macroscopic binding constant likely results from binding to two sites with very different affinities, one in the micromolar range and the other in the millimolar range. Thus, the C-terminal domain appears to also be capable of sensing Ca2+ signals.
J. Biol. Chem, 10.1074/jbc.M112232200
Submitted on December 20, 2001
Revised on May 28, 2002
Accepted on May 28, 2002
Structural independence of the two EF-hand domains of caltractin
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