Functional identification of calcium binding residues in bovine alpha-lactalbumin.
The functional role of previously identified calcium binding residues in alpha-lactalbumin (alpha-LA) was investigated by site-directed mutagenesis. Mutation of D82 to alanine did not effect the binding affinity for calcium, the protein structure, or its function in the lactose synthase assay, suggesting that this aspartate side chain is not essential for calcium binding or structural stabilization. In contrast, mutation of either D87 or D88 to alanine completely eliminated the strong calcium binding and altered alpha-LA as shown by several spectroscopically derived properties such as near- and far-UV CD and intrinsic fluorescence studies. These latter two mutants displayed significantly reduced abilities to stimulate lactose synthase activity (<3.5% of the maximal rate). Additionally, residues K79 and D84, which chelate calcium by backbone carbonyls, were mutated to alanine. K79A lost approximately 50% of its tertiary structure and stability (as determined by CD) but retained full calcium binding activity, indicating that at least the lysine side chain does not influence the carbonyl-mediated calcium coordination. In contrast, D84A lost approximately 25% of its tertiary structure and stability which was accompanied by a modest reduction in calcium affinity. Both mutants were able to stimulate normal lactose synthase activity. The triple mutant, D82A/D87A/D88A alpha-LA, lost its ability to bind calcium, similar to D87A and D88A. These studies clearly demonstrate the importance and variation of side chain interactions, which might be the seminal event in the establishment of the correct calcium binding loop conformation, possibly to stabilization and final folding of the overall protein structure.
Anderson
P J
PJ
Department of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Brooks
C L
CL
Berliner
L J
LJ
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D006801:Humans; D007768:Lactalbumin / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation
D007768:Lactalbumin; D002118:Calcium