Mutating aspartate in the calcium-binding site of α-lactalbumin: Effects on the protein stability and cation binding

Abstract

The residue Asp87, which is in the calcium-binding loop of bovine α-lactalbumin (α-LA) and provides a side-chain carboxylate oxygen for ligand Ca(II) co-ordination, was substituted by either alanine or asparagine. The physical properties and calcium-binding affinities were monitored by intrinsic fluorescence and circular dichroism spectroscopy. D87A α-LA displayed a total loss of rigid tertiary structure, a dramatic loss in secondary structure and negligible calcium affinity [Anderson et al. (1997) Biochemistry, 36, 11648-11654]. On the contrary, D87N α-LA displayed native-like secondary structure with a somewhat destabilized tertiary structure. When the well-documented N-terminal methionine was enzymatically removed from D87N α-LA [Veprintsev et al. (1999) Proteins: Struct. Funct. Genet., 37, 65-72], the structure appeared to more closely resemble native α-LA. Remarkably, the thermal transition mid-temperature of apo-desMetD87N α-LA was ∼31°C versus native apo- α-LA (∼25°C), probably due to negative charge 'compensation' in the calcium co-ordination site. On the other hand, the transition mid-temperature of Ca(II)-bound desMetD87N α-LA was ∼57°C versus native α-LA (∼66°C), which was related to a decreased Ca(II) affinity (K = ∼2.1 × 105 versus ∼1.7 × 107/M at 40°C, respectively). These results reaffirm that alanine substitution in site specific mutagenesis is not always a prudent choice. Substitutions must be conservative with only minimal changes in functional groups and side-chain volume.