Characterization of long and stable de novo single alpha-helix domains provides novel insight into their stability.
Characterization of long and stable de novo single alpha-helix domains provides novel insight into their stability.
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DOI：
10.1038/srep44341
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发表时间：
2017-03-13
影响因子：
4.6
通讯作者：
Peckham M
中科院分区：
文献类型：
--
作者：
Wolny M;Batchelor M;Bartlett GJ;Baker EG;Kurzawa M;Knight PJ;Dougan L;Woolfson DN;Paci E;Peckham M
天然存在的单 α 螺旋 (SAH) 富含 Arg (R)、Glu (E) 和 Lys (K) 残基，并通过多个盐桥稳定。了解盐桥如何提高其稳定性具有挑战性，因为 SAH 很长且序列高度可变。因此，我们设计并测试了包含7个残基重复的简单的从头98个残基多肽（AEEEXXX，其中X是K或R），预期促进Glu和Lys/Arg之间的盐桥形成。富含 Lys 的序列（EK3 (AEEEKKK) 和 EK2R1 (AEEEKRK)）均形成 SAH，其中 EK2R1 更具螺旋性和热稳定性，表明 Arg 增加了稳定性。在天然存在的肌球蛋白 6 SAH 中用精氨酸替代赖氨酸（反之亦然）同样会增加（或降低）其稳定性。然而，富含精氨酸的从头序列（ER3 (AEEERRR) 和 EK1R2 (AEEEKRR)）聚集。将 PDB 分析与分子模型相结合提供了合理的解释，证明 Glu 和 Arg 更常见地形成盐桥，利用更广泛的旋转异构体构象，并且比 Glu-Lys 更具动态性。 Arg 的这种混杂性质有助于解释富含 Arg 的 SAH 从头聚集的倾向增加。重要的是，特定的 K:R 比率可能对于确定从头和天然存在的多肽的螺旋稳定性很重要，从而为单个 α 螺旋如何稳定提供了新的见解。
Naturally-occurring single α-helices (SAHs), are rich in Arg (R), Glu (E) and Lys (K) residues, and stabilized by multiple salt bridges. Understanding how salt bridges promote their stability is challenging as SAHs are long and their sequences highly variable. Thus, we designed and tested simple de novo 98-residue polypeptides containing 7-residue repeats (AEEEXXX, where X is K or R) expected to promote salt-bridge formation between Glu and Lys/Arg. Lys-rich sequences (EK3 (AEEEKKK) and EK2R1 (AEEEKRK)) both form SAHs, of which EK2R1 is more helical and thermo-stable suggesting Arg increases stability. Substituting Lys with Arg (or vice versa) in the naturally-occurring myosin-6 SAH similarly increased (or decreased) its stability. However, Arg-rich de novo sequences (ER3 (AEEERRR) and EK1R2 (AEEEKRR)) aggregated. Combining a PDB analysis with molecular modelling provides a rational explanation, demonstrating that Glu and Arg form salt bridges more commonly, utilize a wider range of rotamer conformations, and are more dynamic than Glu–Lys. This promiscuous nature of Arg helps explain the increased propensity of de novo Arg-rich SAHs to aggregate. Importantly, the specific K:R ratio is likely to be important in determining helical stability in de novo and naturally-occurring polypeptides, giving new insight into how single α-helices are stabilized.