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J. Biol. Chem., Vol. 275, Issue 40, 31178-31182, October 6, 2000

This Article Full Text Full Text (PDF) All Versions of this Article: 275/40/31178    most recent M001622200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nowotny, M. Articles by Kuznicki, J. Search for Related Content PubMed PubMed Citation Articles by Nowotny, M. Articles by Kuznicki, J. Social Bookmarking                      What's this?

Characterization of the Interaction of Calcyclin (S100A6) and Calcyclin-binding Protein^*

Marcin Nowotny, Shibani Bhattacharya^§, Anna Filipek, Andrzej M. Krezel^¶, Walter Chazin^§, and Jacek Kuznicki

From the  Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland and the ^§ Department of Biochemistry and the ^¶ Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37232-0146

Calcyclin (S100A6) is an S100 calcium-binding protein whose expression is up-regulated in proliferating and differentiating cells. A novel 30-kDa protein exhibiting calcium-dependent calcyclin-binding (calcyclin-binding protein, CacyBP) had been identified, purified, and cloned previously (Filipek, A., and Kuznicki, J. (1998) J. Neurochem. 70, 1793-1798). Here, we have defined the calcyclin binding region using limited proteolysis and a set of deletion mutants of CacyBP. A fragment encompassing residues 178-229 (CacyBP-(178-229)) was capable of full binding to calcyclin. CacyBP-(178-229) was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified. The protein fragment cleaved from the glutathione S-transferase fusion protein was shown by CD to contain 5% -helix, 15%  -sheet, and 81% random coil. Fluorescence spectroscopy was used to determine calcyclin dissociation constants of 0.96 and 1.2 µM for intact CacyBP and CacyBP-(178-229), respectively, indicating that the fragment can be used for characterization of calcyclin-CacyBP interactions. NMR analysis of CacyBP-(178-229) binding-induced changes in the chemical shifts of ¹⁵N-enriched calcyclin revealed that CacyBP binding occurs at a discrete site on calcyclin with micromolar affinity.

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