Cooperation between a salt bridge and the hydrophobic core triggers fold stabilization in a Trp-cage miniprotein
Cooperation between a salt bridge and the hydrophobic core triggers fold stabilization in a Trp-cage miniprotein
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DOI：
10.1021/bi701371x
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发表时间：
2008-01-22
期刊：
影响因子：
2.9
通讯作者：
Perczel, Andras
中科院分区：
文献类型：
--
作者：
Hudaky, Peter;Straner, Pal;Perczel, Andras
微蛋白是研究各种蛋白质结构修饰效应如温度、pH、点突变、H-键、盐桥、分子堆积等的适当模型。Tc 5 b，一种20-残基Trp-笼蛋白是具有稳定3D折叠的最小的此类模型之一（Neidigh W.et al.（2002）Nat.Struct.Biol.9，425-430）。然而，Tc 5 b表现出相当大的热敏性，并且仅在相对较低的温度下稳定。在这里，我们报告了一个系统的调查，通过解决其解决方案的结构在不同的环境中，不同的温度和pH值的稳定性的影响Tc 5 b的结构因素。确定的关键相互作用是疏水堆积的芳环Tyr 3和Trp 6和Asp 9和Arg 18之间形成的盐桥。为了验证这些相互作用的重要性，选择的变体（突变，糖基化和截短）的Tc 5 b的设计，制备，并通过NMR研究。事实上，消除任何一种关键相互作用都会使结构高度不稳定。这些观察结果使我们能够设计一种新的变体Tc 6 b，与Tc 5 b的差异仅在于亚甲基，其中两种关键相互作用同时优化。Tc 6 b表现出增强的热稳定性，并在生理温度下采用稳定的折叠。
Miniproteins are adequate models to study various protein-structure modifying effects such as temperature, pH, point mutation(s), H-bonds, salt bridges, molecular packing, etc. Tc5b, a 20-residue Trp-cage protein is one of the smallest of such models with a stable 3D fold (Neidigh J. W.et al. (2002) Nat. Struct. Biol. 9, 425-430). However, Tc5b exhibits considerable heat-sensitivity and is only stable at relatively low temperatures. Here we report a systematic investigation of structural factors influencing the stability of Tc5b by solving its solution structure in different environments, varying temperature, and pH. The key interactions identified are the hydrophobic stacking of the aromatic rings of Tyr3 and Trp6 and the salt bridge formed between Asp9 and Arg18. To verify the importance of these interactions, selected variants (mutated, glycosylated and truncated) of Tc5b were designed, prepared, and investigated by NMR. Indeed, elimination of either of the key interactions highly destabilizes the structure. These observations enabled us to design a new variant, Tc6b, differing only by a methylene group from Tc5b, in which both key interactions are optimized simultaneously. Tc6b exhibits enhanced heat stability and adopts a stable fold at physiological temperature.