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Characterization of calretinin I–II as an EF-hand, Ca²⁺, H⁺-sensing domain

Magorzata Palczewska^1,2, Gyula Batta³, Patrick Groves^1,4, Sara Linse⁵ and Jacek Kunicki^1,6

¹ Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, Poland
² Centro Nacional de Biotecnologia CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain
³ Research Group for Antibiotics of the Hungarian Academy of Sciences and Department of Pharmaceutical Chemistry, University of Debrecen, Debrecen, Hungary
⁴ Centro de Investigaciones Biológicas, Madrid, Spain
⁵ Department of Biophysical Chemistry, University of Lund, Lund, Sweden
⁶ International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland

(RECEIVED January 18, 2005; FINAL REVISION March 29, 2005; ACCEPTED April 11, 2005)

Calretinin, a neuronal protein with well-defined calcium-binding properties, has a poorly defined function. The pH dependent properties of calretinin (CR), the N-terminal (CR I–II), and C-terminal (CR III–VI) domains were investigated. A drop in pH within the intracellular range (from pH 7.5 to pH 6.5) leads to an increased hydrophobicity of calcium-bound CR and its domains as reported by fluorescence spectroscopy with the hydrophobic probe 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). The TNS data for the N- and C-terminal domains of CR are additive, providing further support for their independence within the full-length protein. Our work concentrated on CR I–II, which was found to have hydrophobic properties similar to calmodulin at lower pH. The elution of CR I–II from a phenyl-Sepharose column was consistent with the TNS data. The pH-dependent structural changes were further localized to residues 13–28 and 44–51 using nuclear magnetic resonance spectroscopy chemical shift analysis, and there appear to be no large changes in secondary structure. Protonation of His12 and/or His27 side chains, coupled with calcium chelation, appears to lead to the organization of a hydrophobic pocket in the N-terminal domain. CR may sense and respond to calcium, proton, and other signals, contributing to conflicting data on the proteins role as a calcium sensor or calcium buffer.

Keywords: calcium; calretinin; calbindin D28k; EF-hand; ischemia; pH response

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051369805.

Reprint requests to: Patrick Groves, Centro de Investigaciones Biológicas, Calle Ramiro de Maeztu 9, 28040 Madrid, Spain; e-mail: pdgroves{at}cib.csic.es; fax: +34-915627518.

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