Changes in structure and stability of calbindin-D(28K) upon calcium binding.
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 approximately 10(7.5) and approximately 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.
Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA. venyaminov.sergei@mayo.edu
Sergei Yu
SY
Venyaminov
Elena S
ES
Klimtchuk
Zeljko
Z
Bajzer
Theodore A
TA
Craig
D000818:Animals; D001665:Binding Sites; D001923:Brain Chemistry; D064026:Calbindins; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D064030:S100 Calcium Binding Protein G / Q000737:chemistry* / Q000378:metabolism; D013057:Spectrum Analysis; D013816:Thermodynamics; D017186:Titrimetry
D064026:Calbindins; D064030:S100 Calcium Binding Protein G; D002118:Calcium