Mutating aspartate in the calcium-binding site of alpha-lactalbumin: effects on the protein stability and cation binding.
Mutating aspartate in the calcium-binding site of alpha-lactalbumin: effects on the protein stability and cation binding.
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α-乳清蛋白钙结合位点的天冬氨酸突变：对蛋白质稳定性和阳离子结合的影响。
DOI：
10.1093/protein/14.10.785
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Berliner,LJ
中科院分区：
文献类型：
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作者：
Permyakov,SE;Uversky,VN;Veprintsev,DB;Cherskaya,AM;Brooks,CL;Permyakov,EA;Berliner,LJ
将牛α-乳清蛋白（α-LA）钙结合环中的Asp 87残基用丙氨酸或天冬酰胺取代，Asp 87为配体Ca（II）配位提供侧链羧酸氧。的物理性质和钙结合亲和力进行了监测的固有荧光和圆二色光谱。D87 A α-LA显示刚性三级结构完全丧失，二级结构显著丧失，钙亲和力可忽略不计[Andersonet al.（1997）Biochemistry，36，11648-11654]。而D87 N α-LA的二级结构与天然结构相似，三级结构不稳定。当从D87 N α-LA中酶促除去充分记录的N-末端甲硫氨酸时[Veprintsevet al. 05.《蛋白质：结构功能》（1999）Genet.，37，65-72]，结构似乎更接近天然α-LA。值得注意的是，apo-desMetD 87 N α-LA的热转变中间温度为约31°C，而天然apo- α-LA为约25°C，这可能是由于钙配位位点的负电荷“补偿”。另一方面，与天然α-LA（约66°C）相比，Ca（II）结合desMetD 87 N α-LA的转变中间温度约为57°C，这与Ca（II）亲和力降低有关（40°C时K分别为约2.1× 105和约1.7×107/M）。这些结果再次证实，丙氨酸取代位点特异性诱变并不总是一个谨慎的选择。取代必须是保守的，官能团和侧链体积只有最小的变化。
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 [Andersonet 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 [Veprintsevet 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= ~2.1×105versus ~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.