Changes in structure and stability of calbindin-D 28K upon calcium binding

Abstract

Calbindin-D 28K 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. Ca 2+ titration monitored by fluorescence spectroscopy reveals the presence of two classes of calcium-binding sites with association constants ∼10 7.5 and ∼10 8.9 M −1. CD spectra in the far-UV spectral range show minor changes upon Ca 2+ titration, implying that the secondary structure of calbindin-D 28K 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 Ca 2+ 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.