A modular protein engineering toolkit

天然蛋白质具有令人惊叹的各种功能。合成生物技术发展的一个关键目标是设计不是来自自然序列的新的功能蛋白质。蛋白质设计是一个年轻的领域，从DNA和多肽合成技术开始。最初设计的蛋白质是四螺旋束，稳定得令人惊讶，但没有功能。蛋白质设计还不是常规的，而且有几个限制。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，这种方法计算成本很高，因此仅限于较小的区域。相比之下，高度规则和可预测的DNA结构允许可预测地设计和构建大型“折纸”对象。在此，我们提出了一种模块化蛋白质构建工具包，它使用规则和可扩展的骨架支架，但可以以原子精度插入更小的计算设计功能域。这样的系统将具有高度规则的DNA折纸的一些可组合性和可设计性，并具有计算蛋白质设计的所有优势。该项目从RFR家族中合成的β-螺线管蛋白开始。这个家族有一个5个残基重复，公认的ADLSG。其中四个重复形成一个近乎正方形，这些正方形堆叠形成螺线管。重复序列的A和L残基形成疏水核心，其他残基形成表面有很大的自由度。螺线管核心的宽度与DNA相同，但在化学上更具通用性。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。在这些螺线管中，我们使用一种新的分层无片段技术，插入了经过计算设计的环，以及整个蛋白质结构域(MacDonald等人。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到一个多部分的模块化蛋白质设计工具包中。我们将使用功能循环来扩展脚手架，并设计接口以实现更高级别结构的模块化建筑。该项目将涉及计算蛋白质设计、蛋白质特性和结构生物学。

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.