Engineering subunit association of multisubunit proteins: A dimeric streptavidin
Engineering subunit association of multisubunit proteins: A dimeric streptavidin
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DOI：
10.1073/pnas.94.12.6153
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发表时间：
1997-06-10
影响因子：
11.1
通讯作者：
Cantor, CR
中科院分区：
文献类型：
--
作者：
Sano, T;Vajda, S;Cantor, CR
已经通过使用有效结合自由能计算的分子建模设计了二聚链霉亲和素，所述有效结合自由能计算将结合自由能分解为静电、去溶剂化和侧链熵损失项。组氨酸-127->天冬氨酸（H127 D）突变足以在亚基之间引入静电排斥，从而防止天然四聚体的形成。然而，二聚体-二聚体界面的高疏水性（其将暴露于二聚体链霉亲和素中的溶剂）表明所得分子在水性介质中具有非常低的溶解度。与计算一致，含有H127 D突变的链霉亲和素形成不溶性聚集体。因此，主要的设计目标是在保持基本结构的同时降低二聚体-二聚体界面的疏水性，自由能计算表明，二聚体-二聚体界面的疏水性可以通过删除从G113到W120的环而显著降低，所述环应该与二聚体分子中的生物素没有明显的接触。含有H127 D突变和环缺失的菌株在生物素存在下形成可溶性二聚链霉亲和素。
A dimeric streptavidin has been designed by molecular modeling using effective binding free energy calculations that decompose the binding free energy into electrostatic, desolvation, and side chain entropy loss terms, A histidine-127 --> aspartic acid (H127D) mutation was sufficient to introduce electrostatic repulsion between subunits that prevents the formation of the natural tetramer. However, the high hydrophobicity of the dimer-dimer interface, which would be exposed to solvent in a dimeric streptavidin, suggests that the resulting molecule would have very low solubility in aqueous media, In agreement with the calculations, a streptavidin containing the H127D mutation formed insoluble aggregates, Thus, the major design goal was to reduce the hydrophobicity of the dimer-dimer interface while maintaining the fundamental structure, Free energy calculations suggested that the hydrophobicity of the dimer-dimer interface could be reduced significantly by deleting a loop from G113 through W120 that should have no apparent contact with biotin in a dimeric molecule, The resulting protein, containing both the H127D mutation and the loop deletion, formed a soluble dimeric streptavidin in the presence of biotin.