Changes in structure and stability of calbindin-D28K upon calcium binding

Sergei Yu Venyaminov, Elena S. Klimtchuk, Zeljko Bajzer, Theodore A. Craig

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

Calbindin-D28K is a biologically important protein required for normal neural function and for the transport of calcium in epithelial cells of the intestine and kidney. We have used fluorescence and circular dichroism (CD) spectroscopy to characterize the effects of calcium binding on the structure and stability of calbindin. Ca2+ titration monitored by fluorescence spectroscopy reveals the presence of two classes of calcium-binding sites with association constants ∼107.5 and ∼108.9 M -1. CD spectra in the far-UV spectral range show minor changes upon Ca2+ titration, implying that the secondary structure of calbindin-D28K is not greatly affected. On the basis of the CD spectra in the near-UV spectral range, we conclude that the tertiary structure is more sensitive to Ca2+ addition. The most significant change occurs between pCa 7.0 and pCa 8.0. The variations in the protein thermostability are correlated with those in the near-UV CD spectra. The enthalpy changes upon heat denaturation of calbindin in the apo-state are characteristic of proteins containing several weakly interacting domains with similar thermodynamical properties. Thus, calcium binding by calbindin-D 28K largely affects the local structure around the aromatic residues and the thermal stability of the protein; the changes in the secondary structure are insignificant.

Original languageEnglish (US)
Pages (from-to)97-105
Number of pages9
JournalAnalytical Biochemistry
Volume334
Issue number1
DOIs
StatePublished - Nov 1 2004

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Keywords

  • Far-UV circular dichroism spectra
  • Fluorescence
  • Near-UV circular dichroism spectra
  • Secondary structure
  • Temperature denaturation

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Venyaminov, S. Y., Klimtchuk, E. S., Bajzer, Z., & Craig, T. A. (2004). Changes in structure and stability of calbindin-D28K upon calcium binding. Analytical Biochemistry, 334(1), 97-105. https://doi.org/10.1016/j.ab.2004.07.032