Leng Xiyue, Albanese Katherine I, Golub Lia R, Norman Arthur A, Clayden Jonathan, Woolfson Derek N
School of Chemistry, University of Bristol Cantock's Close Bristol BS8 1TS UK
Max Plack-Bristol Centre for Minimal Biology, University of Bristol Cantock's Close Bristol BS8 1TS UK.
Chem Sci. 2025 Aug 27. doi: 10.1039/d5sc05576h.
Computational protein design is advancing rapidly. However, approaches and methods are needed to increase success rates and to elaborate designs. Here we describe the combination of rational and computational design to deliver three-helix bundle (3HB) peptide assemblies and single-chain proteins with control over topology and thermal stability. First, we garner sequence-to-structure relationships from antiparallel 3HBs in the Protein Data Bank. This gives core-packing rules, including layers of hydrogen-bonded polar residues, which are combined with surface-charge patterning to design complementary sequences for acidic (A), basic (B), and neutral (N) helices. By altering the design of the N helix, two sets of synthetic peptides are generated for clockwise and anticlockwise arrangements of the three-helix assemblies. Solution-phase characterisation shows that both ABN peptide mixtures form stable, heterotrimeric assemblies consistent with the targeted 'up-down-up' topologies. Next, AlphaFold2 models for both designs are used to seed computational designs of single-chain proteins where the helices are connected by loop building. Synthetic genes for these express in to yield soluble, monomeric, and thermally stable proteins. By systematically introducing additional polar layers within the core, the thermal stability of these proteins is varied without compromising the specificity of the helix-helix interactions. Chemical and thermal denaturation reveals comparable thermodynamic parameters to those of highly stable natural proteins. Four X-ray crystal structures confirm that the design models and AlphaFold2 predictions match to sub-Å accuracy.
计算蛋白质设计正在迅速发展。然而，需要一些方法和手段来提高成功率并完善设计。在此，我们描述了合理设计与计算设计相结合的方法，以构建具有拓扑结构和热稳定性可控的三螺旋束（3HB）肽组装体和单链蛋白质。首先，我们从蛋白质数据库中的反平行3HB中获取序列与结构的关系。这给出了核心堆积规则，包括氢键连接的极性残基层，这些规则与表面电荷模式相结合，用于设计酸性（A）、碱性（B）和中性（N）螺旋的互补序列。通过改变N螺旋的设计，生成了两组用于三螺旋组装体顺时针和逆时针排列的合成肽。溶液相表征表明，两种ABN肽混合物均形成了与目标“上-下-上”拓扑结构一致的稳定异源三聚体组装体。接下来，两种设计的AlphaFold2模型被用于作为单链蛋白质计算设计的种子，其中螺旋通过环构建连接。这些蛋白质的合成基因在大肠杆菌中表达，产生可溶性、单体且热稳定的蛋白质。通过在核心区域系统地引入额外的极性层，可以改变这些蛋白质的热稳定性，而不影响螺旋-螺旋相互作用的特异性。化学和热变性实验揭示了与高度稳定的天然蛋白质相当的热力学参数。四个X射线晶体结构证实，设计模型与AlphaFold2预测的匹配精度达到亚埃级。
