A Basis Set of de Novo Coiled-Coil Peptide Oligomers for Rational Protein Design and Synthetic Biology
A Basis Set of de Novo Coiled-Coil Peptide Oligomers for Rational Protein Design and Synthetic Biology
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DOI：
10.1021/sb300028q
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发表时间：
2012-06-01
影响因子：
4.7
通讯作者：
Woolfson, Derek N.
中科院分区：
文献类型：
--
作者：
Fletcher, Jordan M.;Boyle, Aimee L.;Woolfson, Derek N.
蛋白质工程、化学生物学和合成生物学将受益于肽和蛋白质成分的工具包，这些工具包可以在系统之间可靠地交换，同时保持其结构和功能的完整性。理想情况下，这些组件应该在序列、结构、稳定性、相互作用和功能的所有方面都是高度定义和可预测的。为了建立这样一个工具包，在这里，我们提出了一套基础的从头设计的α-螺旋卷曲螺旋肽，采用定义和良好的特点，平行二聚体，三聚体和四聚体状态。这些设计是基于文献和已知螺旋线圈X射线晶体结构数据库的分析中的序列与结构的关系。这些给出前景序列以指定靶向寡聚体状态。设计过程的一个关键特征是，这些位点之外的序列位置被认为对结构特异性是非必需的;因此，它们被称为背景，保持非描述性，并可用于稍后需要的突变。合成肽的特点是在溶液中的圆二色性光谱和分析超离心，并通过X射线晶体学确定其结构。有趣的是，迄今为止广泛使用的卷曲螺旋二聚体规格的经验经验法则在设计的系统中并不成立。然而，所需的寡聚状态是通过数据库通知的特定前景的重新设计实现的，并通过实验证实。我们设想该基组将用于指导和控制蛋白质组装，并在化学和合成生物学中具有潜在应用。为了帮助这样的努力，我们介绍Pcomp，在线注册表的肽组分的蛋白质设计和合成生物学应用。
Protein engineering, chemical biology, and synthetic biology would benefit from toolkits of peptide and protein components that could be exchanged reliably between systems while maintaining their structural and functional integrity. Ideally, such components should be highly defined and predictable in all respects of sequence, structure, stability, interactions, and function. To establish one such toolkit, here we present a basis set of de novo designed alpha-helical coiled-coil peptides that adopt defined and well-characterized parallel dimeric, trimeric, and tetrameric states. The designs are based on sequence-to-structure relationships both from the literature and analysis,, of a database of known coiled-coil X-ray crystal structures. These give foreground sequences to specify the targeted oligomer state. A key feature of the design process is that sequence positions outside of these sites are considered non-essential for structural specificity; as such, they are referred to as the background, are kept non-descript, and are available for mutation as required later. Synthetic peptides were characterized in solution by circular-dichroism spectroscopy and analytical ultracentrifugation, and their structures were determined by X-ray crystallography. Intriguingly, a hitherto widely used empirical rule-of-thumb for coiled-coil dimer specification does not hold in the designed system. However, the desired oligomeric state is achieved by database-informed redesign of that particular foreground and confirmed experimentally. We envisage that the basis set will be of use in directing and controlling protein assembly, with potential applications in chemical and synthetic biology. To help with such endeavors, we introduce Pcomp, an on-line registry of peptide components for protein-design and synthetic-biology applications.