Protein thermal stability enhancement by designing salt bridges: a combined computational and experimental study.
Protein thermal stability enhancement by designing salt bridges: a combined computational and experimental study.
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DOI：
10.1371/journal.pone.0112751
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Tseng CP
中科院分区：
文献类型：
--
作者：
Lee CW;Wang HJ;Hwang JK;Tseng CP
蛋白质热稳定性是医学和工业应用中考虑的重要因素。许多与蛋白质热稳定性相关的结构特征已被阐明，增加盐桥被认为是提高蛋白质热稳定性的最有效策略之一。然而，盐桥的准确模拟仍然很困难。在这项研究中，提出了一种新的盐桥设计方法的基础上，统计分析的10,556个表面盐桥的6,493个X射线蛋白质结构。这些盐桥首先根据配对残基、二级结构位置和Cα-Cα距离进行分类。从统计分析中归纳出的配对偏好被用来构建盐桥配对指数，并用于加权静电吸引模型中，以找到设计盐桥的有效配对。该模型还加上B-因子，加权接触数，相对溶剂的可及性，和保护预筛选，以确定适当的盐桥的热适应性设计的残留物。根据我们的方法，在嗜温β-葡萄糖苷酶上设计了8个假定的盐桥，并构建了24个变体来验证预测。六种盐桥的存在导致了酶热稳定性的提高。检测到推定盐桥N437 K-D49、E96 R-D28、E96 K-D28、S440 K-E70、T231 K-D388和Q277 E-D282的熔融温度分别显著增加8.8、4.8、3.7、1.3、1.2和0.7°C。将T231 K-D388的极性反转为T231 D-D388 K导致解链温度进一步增加3.6°C，这可能是由亚基内静电相互作用转变为亚基间静电相互作用（取决于局部环境）引起的。热稳定变体（N437 K、E96 R、T231 D和D388 K）的组合产生15.7°C的解链温度增加。因此，本研究展示了一种新的方法，盐桥的热适应性设计，通过推断合适的位置和替代。
Protein thermal stability is an important factor considered in medical and industrial applications. Many structural characteristics related to protein thermal stability have been elucidated, and increasing salt bridges is considered as one of the most efficient strategies to increase protein thermal stability. However, the accurate simulation of salt bridges remains difficult. In this study, a novel method for salt-bridge design was proposed based on the statistical analysis of 10,556 surface salt bridges on 6,493 X-ray protein structures. These salt bridges were first categorized based on pairing residues, secondary structure locations, and Cα–Cα distances. Pairing preferences generalized from statistical analysis were used to construct a salt-bridge pair index and utilized in a weighted electrostatic attraction model to find the effective pairings for designing salt bridges. The model was also coupled with B-factor, weighted contact number, relative solvent accessibility, and conservation prescreening to determine the residues appropriate for the thermal adaptive design of salt bridges. According to our method, eight putative salt-bridges were designed on a mesophilic β-glucosidase and 24 variants were constructed to verify the predictions. Six putative salt-bridges leaded to the increase of the enzyme thermal stability. A significant increase in melting temperature of 8.8, 4.8, 3.7, 1.3, 1.2, and 0.7°C of the putative salt-bridges N437K–D49, E96R–D28, E96K–D28, S440K–E70, T231K–D388, and Q277E–D282 was detected, respectively. Reversing the polarity of T231K–D388 to T231D–D388K resulted in a further increase in melting temperatures by 3.6°C, which may be caused by the transformation of an intra-subunit electrostatic interaction into an inter-subunit one depending on the local environment. The combination of the thermostable variants (N437K, E96R, T231D and D388K) generated a melting temperature increase of 15.7°C. Thus, this study demonstrated a novel method for the thermal adaptive design of salt bridges through inference of suitable positions and substitutions.
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DOI：
10.1073/pnas.0808220106
发表时间：
2009-02-24
影响因子：
11.1
作者：
Gribenko, Alexey V.;Patel, Mayank M.;Makhatadze, George I.
通讯作者：
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影响因子：
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