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Protein Engineering, Vol. 14, No. 10, 785-789, October 2001
© 2001 Oxford University Press

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

Sergei E. Permyakov1, Vladimir N. Uversky1,2, Dmitry B. Veprintsev1, Alexandra M. Cherskaya1, Charles L. Brooks3, Eugene A. Permyakov1 and Lawrence J. Berliner4,5,6

1 Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region 142290, Russia, 2 Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, 3 Departments of Veterinary Biosciences and Biochemistry and 4 Departments of Chemistry and Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210, USA

The residue Asp87, which is in the calcium-binding loop of bovine {alpha}-lactalbumin ({alpha}-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 {alpha}-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 {alpha}-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 {alpha}-LA [Veprintsev et al. (1999) Proteins: Struct. Funct. Genet., 37, 65–72], the structure appeared to more closely resemble native {alpha}-LA. Remarkably, the thermal transition mid-temperature of apo-desMetD87N {alpha}-LA was ~31°C versus native apo- {alpha}-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 {alpha}-LA was ~57°C versus native {alpha}-LA (~66°C), which was related to a decreased Ca(II) affinity (K = ~2.1x105 versus ~1.7x107/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.


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