Building a new type of protein from scratch: functional 310 assemblies

螺旋结构在自然界中广泛用于构建高阶组件和存储信息。这主要是由于它们能够很好地装入高度定义的构象空间。在这些螺旋分子中，多肽α螺旋是最常见的结构，200多万个残基中有33%位于其中。它是生物学的基本常数之一，在蛋白质设计、蛋白质工程和生物技术中广泛用作支架。α螺旋构象高度明确，主链扭转范围窄，螺旋参数紧凑，残基i和残基i+4之间有稳定的螺旋内氢键主链。换句话说，阿尔法螺旋位于一个深而窄的自由能井中。这就是为什么存在于α螺旋区域周围的替代结构在自然界中不那么普遍的原因。它们位于这口井的边缘，因此热力学上不太稳定。其中一种替代组件是310螺旋。310这个名字源于每转3个氨基酸（而不是在alpha中观察到的3.6个）和一个螺旋内氢键循环中的10个原子。与α螺旋相比，在相同的残基数下形成了更紧密、更长的螺旋结构，氢键模式为i到i+3。虽然它们实际上比α螺旋更早被发现，但在水介质中构建三级或四级结构时，大自然似乎忽略了它们。因此，我们感兴趣的是研究为什么大自然没有在进化中发现和利用310，以及它们是否仍然可以存在或重新设计。伍尔夫森小组最近的工作已经证实了八聚体310螺旋束的溶液状态和晶体结构。每个组成螺旋有三种类型的氨基酸具有特定的作用：(1)疏水亮氨酸残基指向组装的中心，以实现螺旋-螺旋稳定。(2)静电残基对Glu和Lys，通过盐桥接增加水溶性，进一步稳定组装。(3)季胺基酸有利于更紧密的螺旋旋转。有趣的是，310基的四元结构可以可靠地形成，但只有当包含α， α -二取代氨基酸（如Aib）时，由于α碳上更大的空间约束引入了更尖锐的螺旋转向。因此，我们很有兴趣通过将合成氨基酸、季氨基酸和极性氨基酸纳入肽序列来多样化促进310的合成氨基酸调色板。在此之前，Clayden小组通过暂时形成咪唑烷酰脲的第二个立体中心证明了氨基酸的对映选择性α -芳基化策略。这种四元α芳基化氨基酸在不破坏螺旋折叠的情况下插入，并使用对映不纯氨基酸前体作为不对称的来源。基于310螺旋的四级结构不会自然地出现在长链中，设计和功能化它们可以使我们探索新的化学空间，超越传统的α螺旋。因此，我们的目标是探索其他序列使用从头设计和纳入非天然季氨基酸。

Helical structures are used extensively in nature to build high-order assemblies and store information. This is largely due to their ability to pack well into highly defined conformational space. Among such helical molecules, the polypeptide alpha helix is the most commonly seen structure, within which 33% of the over 2 million residues are located. It is one of the fundamental constants of biology and widely used as a scaffold in protein design, protein engineering, and biotechnology. Alpha helices are conformationally highly defined with narrow range of backbone torsion and tight helical parameters, as well as the stabilising intrahelical hydrogen-bonding backbone between residues i and i+4. In another word, the alpha helix sits in a deep and narrow free-energy well. This is the reason why alternative structures that exist around the alpha-helical region are less prevalent in nature. They lie on the rim of this well and are therefore thermodynamically less stable. One of such alternative assemblies is the 310 helices. The name 310 originates from the 3 amino acids per turn (instead of 3.6 observed in alpha) and the 10 atoms within one intrahelical H-bond cycle. It forms a tighter and longer helical structure for the same number of residues in comparison to the alpha helix, with H-bond pattern i to i+3. Although they were in fact discovered earlier than alpha helices, nature seems to have overlooked them when building tertiary or quaternary structure in aqueous media. Therefore, it is of our interest to investigate why nature has not found and exploited 310s evolutionarily and whether they can nonetheless exist or be designed de novo. Recent work in the Woolfson group has confirmed the solution-state and crystal structure of an octameric 310 helix bundle. Each constituent helix has three types of amino acids with specific roles: (1) Hydrophobic Leu residues that point into the centre of the assembly to achieve helix-helix stabilisation. (2) Electrostatic residue pairs, Glu and Lys, to increase water solubility and further stabilise the assembly through salt bridging. (3) Quaternary amino acid to favour the tighter helical turns. Interestingly, 310-based quaternary structures can form reliably but only when incorporating alpha,alpha-disubstituted amino acids (such as Aib) due to the sharper helical turn introduced by greater steric constraints at the alpha carbon. Therefore, we are intrigued to diversify the 310-promoting synthetic amino acids palette by incorporating synthetic, quaternary, and polar amino acids into the peptide sequence. Previously, the Clayden group demonstrated an enantioselective alpha-arylation of amino acids strategy via the temporary formation of a second stereogenic centre of imidazolidinyl urea. Such quaternary alpha-arylated amino acids were shown to be inserted without disrupting the helical folding and uses enantiopure amino acid precursors as the source of asymmetry. 310 helix based quaternary structures do not occur naturally in long chains, designing and functionalising them could enable us to explore new chemical space that go beyond what was achieved with traditional alpha helices. Thus, our goal is to explore other sequences using de novo design and incorporate unnatural quaternary amino acids.