Fletcher Jordan M, Boyle Aimee L, Bruning Marc, Bartlett Gail J, Vincent Thomas L, Zaccai Nathan R, Armstrong Craig T, Bromley Elizabeth H C, Booth Paula J, Brady R Leo, Thomson Andrew R, Woolfson Derek N
School of Chemistry, University of Bristol, Cantocks Close, Bristol BS8 1TS, UK.
ACS Synth Biol. 2012 Jun 15;1(6):240-50. doi: 10.1021/sb300028q. Epub 2012 May 14.
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 α-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.
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组件的工具包，这些组件能够在不同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，这些组件在序列、结构、稳定性、相互作用和功能的所有方面都应具有高度的确定性和可预测性。为了建立这样一个工具包，我们在此展示了一组从头设计的α-螺旋卷曲螺旋肽的基础集，这些肽能够形成确定的、特征明确的平行二聚体、三聚体和四聚体状态。这些设计基于文献中的序列与结构关系以及对已知卷曲螺旋X射线晶体结构数据库的分析。这些提供了前景序列以指定目标寡聚体状态。设计过程的一个关键特征是，这些位点之外的序列位置对于结构特异性被认为是非必需的；因此，它们被称为背景，保持不明确，并可根据需要在以后进行突变。合成肽通过圆二色光谱和分析超速离心在溶液中进行表征，其结构通过X射线晶体学确定。有趣的是，一种迄今广泛使用的用于卷曲螺旋二聚体指定的经验法则在设计系统中并不成立。然而，通过对该特定前景进行基于数据库的重新设计并通过实验得到了所需的寡聚体状态。我们设想这个基础集将用于指导和控制蛋白质组装，在化学生物学和合成生物学中具有潜在应用。为了帮助进行此类工作，我们引入了Pcomp，这是一个用于蛋白质设计和合成生物学应用的肽组件在线注册库。
