Mutating aspartate in the calcium-binding site of alpha-lactalbumin: effects on the protein stability and cation binding.
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 approximately 31 degrees C versus native apo- alpha-LA (approximately 25 degrees 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 approximately 57 degrees C versus native alpha-LA (approximately 66 degrees C), which was related to a decreased Ca(II) affinity (K = approximately 2.1 x 10(5) versus approximately 1.7 x 10(7)/M at 40 degrees 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.
Permyakov
S E
SE
Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow region 142290, Russia.
Uversky
V N
VN
Veprintsev
D B
DB
Cherskaya
A M
AM
Brooks
C L
CL
Permyakov
E A
EA
Berliner
L J
LJ
D000818:Animals; D001216:Asparagine / Q000502:physiology; D001224:Aspartic Acid / Q000502:physiology*; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D003001:Cloning, Molecular; D007768:Lactalbumin / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D013050:Spectrometry, Fluorescence; D013696:Temperature
D001224:Aspartic Acid; D001216:Asparagine; D007768:Lactalbumin