A modular protein engineering toolkit

天然蛋白质具有惊人的功能。合成生物学技术发展的一个关键目标是设计新的功能蛋白质，而不是从天然序列中衍生出来。蛋白质设计是一个年轻的领域，始于DNA和肽合成技术。第一个设计的蛋白质是四螺旋束，令人惊讶的稳定，但没有功能。蛋白质设计还不是常规的，并且有一些限制。大多数成功的设计方法在全原子力场中使用势能优化方法，这在计算上是昂贵的，因此仅限于较小的域。相比之下，DNA的高度规则和可预测的结构允许大的“折纸”物体被可预测地设计和构建。在这里，我们提出了一个模块化的蛋白质构建工具包，它使用规则和可扩展的骨架支架，但可以有更小的计算设计的功能域插入原子精度。这样的系统将具有高度规则的DNA折纸的可组合性和可设计性，以及计算蛋白质设计的所有优点。该项目从RFR家族中的合成β螺线管蛋白开始。该家族具有5个残基重复，共有ADLSG。这些重复中的四个形成近正方形，并且这些正方形堆叠以形成螺线管。重复序列的A和L残基形成疏水核心，并且对于形成表面的其他残基存在很大的自由度。螺线管核心的宽度和DNA差不多，但在化学上用途更广。我们已经合成了长度可变的螺线管蛋白质，其重复序列来自残基频率表。我们使用一种新的分层无片段技术，在这些DNA中插入了计算设计的环和整个蛋白质结构域（MacDonald et al. 2016）。这项工作定义了一个可扩展的蛋白质支架部分，我们现在希望将其扩展为多部分模块化蛋白质设计工具包。我们将用功能循环来扩展脚手架，并设计接口以实现更高阶结构的模块化构建。该项目将涉及计算蛋白质设计、蛋白质表征和结构生物学。

Natural proteins perform an amazing repertoire of functions. A key goal of synthetic biology technology development is to design new functional proteins that are not derived from natural sequences. Protein design is a young field, beginning with DNA and peptide synthesis technologies. The first designed proteins were four-helix bundles, surprisingly stable, but not functional. Protein design is not yet routine, and has several limitations. Most successful design methods use a potential energy optimisation approach in an all-atom forcefield, which is computationally expensive and therefore limited to smaller domains. In contrast, the highly regular and predictable structure of DNA allows large "origami" objects to be predictably designed and constructed.Here, we propose a modular protein construction kit that uses regular and extendable backbone scaffolds, but can have smaller computationally designed functional domains inserted with atomic precision. Such a system would have some of the composability and designability of highly regular DNA origami, with all of the advantages of computational protein design. The project begins with synthetic beta-solenoid proteins in the RFR family. This family has a 5 residue repeat, consensus ADLSG. Four of these repeats form a near-square and these squares stack to form the solenoid. The A and L residues of the repeat form the hydrophobic core, and there is a great deal of freedom for the other residues which form the surface. The solenoid core is about the same width as DNA, but much more chemically versatile.We have made synthetic solenoid proteins of variable lengths with repeat sequences drawn from a residue frequency table. Into these solenoids we have inserted computationally designed loops, using a novel hierarchical fragment-free technique, and entire protein domains (MacDonald et al. 2016). This work defined an extendable protein scaffold part, which we now wish to extend into a multi-part modular protein design toolkit. We will extend the scaffold with functional loops, and design interfaces to enable the modular building of higher order structures. The project will involve computational protein design, protein characterization and structural biology.