Protein stabilization by removal of unsatisfied polar groups: Computational approaches and experimental tests
Protein stabilization by removal of unsatisfied polar groups: Computational approaches and experimental tests
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DOI：
10.1021/bi9605191
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发表时间：
1996-06-18
期刊：
影响因子：
2.9
通讯作者：
Tidor, B
中科院分区：
文献类型：
--
作者：
Hendsch, ZS;Jonsson, T;Tidor, B
在一种ARE抑制物(MYL)中，极性和带电侧链在部分埋藏的蛋白质环境中的作用已经被探讨，在MYL中，Met31、Tyr36和Leu40之间的疏水相互作用取代了Arg31、Glu36和Arg40之间的野生型盐桥相互作用。在没有这个盐桥三联体的情况下，通过连续静电计算发现，另外三条侧链在折叠过程中遭受的去溶惩罚比在折叠状态下有利的静电相互作用中恢复的要大。这些侧链(Asn29、Ser44和Glu48)在锰背景下被单独和共同突变为丙氨酸，所产生的突变蛋白的热力学稳定性提高了0.1~1.3kcal/mol的二聚体。近紫外圆二色谱分析表明，所有突变体均表现为协同热熔融，且疏水核心排列紧密，表明保持了构象特异性。用非极性基团取代整个六个残基的ARE变异体(MYL-NA29/SA44/EA48)的二聚体稳定性比野生型高5.1千卡/摩尔，表明用疏水残基取代掩埋或部分掩埋的带电和极性侧链的策略可以导致实质性的稳定。
The role of polar and charged side chains in partially buried protein environments has been probed in a variant of Are repressor (MYL) in which hydrophobic interactions between Met31, Tyr36, and Leu40 replace the wild-type salt-bridge interactions between Arg31, Glu36, and Arg40. In the absence of this salt-bridge triad, three additional side chains were identified by continuum electrostatic calculations as incurring larger desolvation penalties during folding than were recovered in favorable electrostatic interactions in the folded state. These side chains (Asn29, Ser44, and Glu48) were mutated singly and collectively to alanine in the Mn background, and the thermodynamic stabilities of the resulting mutant proteins were found to be increased by 0.1 to 1.3 kcal/mol of dimer. All of the mutants displayed cooperative thermal melts and appeared to have well-packed hydrophobic cores by near-UV circular dichroism spectroscopy, indicating that conformational specificity is maintained. The Are variant (MYL-NA29/SA44/EA48) in which the entire six-residue polar network is replaced by nonpolar groups is 5.1 kcal/mol of dimer more stable than wild type, indicating that the strategy of replacing buried or partially buried charged and polar side chains with hydrophobic residues can lead to substantial stabilization.