Mutating Aspartate in The Calcium-binding Site of α-lactalbumin: Effects on The Protein Stability and Cation Binding
Document Type
Article
Publication Date
2001
Digital Object Identifier (DOI)
https://doi.org/10.1093/protein/14.10.785
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 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.
Was this content written or created while at USF?
Yes
Citation / Publisher Attribution
Protein Engineering, Design and Selection, v. 14, issue 10, p. 785-789
Scholar Commons Citation
Permyakov, Sergei E.; Uversky, Vladimir N.; Veprintsev, Dmitry B.; Cherskaya, Alexandra M.; rooksHolt, Charles L.; Permyakov, Eugene A.; and Berliner, Lawrence J., "Mutating Aspartate in The Calcium-binding Site of α-lactalbumin: Effects on The Protein Stability and Cation Binding" (2001). Molecular Medicine Faculty Publications. 679.
https://digitalcommons.usf.edu/mme_facpub/679
