Stabilization of an immunoglobulin fold domain by an engineered disulfide bond at the buried hydrophobic region
Stabilization of an immunoglobulin fold domain by an engineered disulfide bond at the buried hydrophobic region
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DOI：
10.1074/jbc.m707078200
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发表时间：
2007-12-14
影响因子：
4.8
通讯作者：
Uegaki, Koichi
中科院分区：
文献类型：
--
作者：
Hagihara, Yoshihisa;Mine, Shouhei;Uegaki, Koichi
我们首次报道了免疫球蛋白折叠结构域通过工程二硫键的稳定性。在以人绒毛膜促性腺激素为特异性抗原的羊驼单域抗体中，Ala（49）和Ile（70）被埋没在结构中。在该位置添加人工二硫键的突变体，其热展开的中点温度比没有添加额外二硫键的突变体高10℃。修饰结构域表现出与野生型结构域相当的抗原结合亲和力。Ala（49）和Ile（70）在骆驼和羊驼单域抗体框架中是保守的。因此，针对不同抗原的结构域有望通过本文研究的工程二硫键来稳定。热力学分析表明，除了环约束的影响外，内部相互作用和水合作用也控制了含二硫键结构域的稳定性。由突变引起的物理性质的变化通常会对这些相互作用产生不可预测和不稳定的影响。在疏水区域引入疏水胱氨酸可以保持蛋白质的疏水性，并有望将不利的突变效应降至最低。
We report for the first time the stabilization of an immunoglobulin fold domain by an engineered disulfide bond. In the llama single-domain antibody, which has human chorionic gonadotropin as its specific antigen, Ala(49) and Ile(70) are buried in the structure. A mutant with an artificial disulfide bond at this position showed a 10 degrees C higher midpoint temperature of thermal unfolding than that without the extra disulfide bond. The modified domains exhibited an antigen binding affinity comparable with that of the wild-type domain. Ala(49) and Ile(70) are conserved in camel and llama single-domain antibody frameworks. Therefore, domains against different antigens are expected to be stabilized by the engineered disulfide bond examined here. In addition to the effect of the loop constraints in the unfolded state, thermodynamic analysis indicated that internal interaction and hydration also control the stability of domains with disulfide bonds. The change in physical properties resulting from mutation often causes unpredictable and destabilizing effects on these interactions. The introduction of a hydrophobic cystine into the hydrophobic region maintains the hydrophobicity of the protein and is expected to minimize the unfavorable mutational effects.