title: Characterization of calretinin I-II as an EF-hand, Ca2+, H+-sensing domain.
abstract: 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 His 12 and/or His 27 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.
authors.affiliation: Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, Poland.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Palczewska
authors.forename: Gyula
authors.initials: G
authors.lastname: Batta
authors.forename: Patrick
authors.initials: P
authors.lastname: Groves
authors.forename: Sara
authors.initials: S
authors.lastname: Linse
authors.forename: Jacek
authors.initials: J
authors.lastname: Kuznicki
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D064032:Calbindin 2; D002118:Calcium / Q000378:metabolism*; D020832:EF Hand Motifs; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010843:Pichia / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011522:Protons; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D064032:Calbindin 2; D011522:Protons; D011993:Recombinant Fusion Proteins; D064030:S100 Calcium Binding Protein G; D005982:Glutathione Transferase; D002118:Calcium