Purification, calcium-binding properties, and conformational studies on a 28-kDa cholecalcin-like protein from bovine brain.
A large-scale preparation method for bovine brain 28-kDa cholecalcin-like protein is described. Flow dialysis binding studies revealed that the protein binds at least 3 mol of Ca2+/mol of protein. The protein undergoes conformational changes on binding calcium as shown by UV differential absorption spectroscopy, near and far UV circular dichroism, and intrinsic fluorescence. Circular dichroism (CD) studies in the far UV indicate an apparent increase in helical content in the presence of Ca2+. The effect of calcium on the protein structure is nearly maximum for 1 Ca2+ bound/protein molecule. UV differential absorption studies on the binding of the Ca2+ agonist Tb3+ and Tb3+ luminescence induced by energy Trp----Tb3+ transfer indicate that Tb3+ binds to two higher affinity Ca2+-binding sites. These sites are probably very close to the single Trp residue. Analysis of the fluorescence parameters of the single tryptophan residue in the apoprotein and its accessibility to ionic and neutral quenchers suggests that this residue is located in a highly hydrophobic domain on the protein surface.
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000302:isolation & purification*; D002417:Cattle; D002942:Circular Dichroism; D003429:Cross Reactions; D007106:Immune Sera; D007413:Intestinal Mucosa / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D064030:S100 Calcium Binding Protein G / Q000302:isolation & purification* / Q000378:metabolism; D013045:Species Specificity; D013050:Spectrometry, Fluorescence
D002135:Calcium-Binding Proteins; D007106:Immune Sera; D064030:S100 Calcium Binding Protein G; D002118:Calcium