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

• Published in: Protein engineering 2001-10, Vol.14 (10), p.785-789
• Main Authors: Permyakov, Sergei E., Uversky, Vladimir N., Veprintsev, Dmitry B., Cherskaya, Alexandra M., Brooks, Charles L., Permyakov, Eugene A., Berliner, Lawrence J.
• Format: Article
• Language: English
• Subjects: a-Lactalbumin; Animals; Asparagine - physiology; Aspartic Acid - physiology; Binding Sites; calcium binding; Cattle; Circular Dichroism; Cloning, Molecular; fluorescence; Lactalbumin - genetics; Lactalbumin - metabolism; Mutation; Spectrometry, Fluorescence; Temperature; thermal stability; α-lactalbumin
• ISSN: 0269-2139; 1741-0126; 1460-213X; 1741-0134
• DOI: 10.1093/protein/14.10.785

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 de-stabilized 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.