Computational protein design is advancing rapidly. Here we describe efficient routes starting from validated parallel and antiparallel peptide assemblies to design two families of α-helical barrel proteins with central channels that bind small molecules. Computational designs are seeded by the sequences and structures of defined de novo oligomeric barrel-forming peptides, and adjacent helices are connected by loop building. For targets with antiparallel helices, short loops are sufficient. However, targets with parallel helices require longer connectors; namely, an outer layer of helix–turn–helix–turn–helix motifs that are packed onto the barrels. Throughout these computational pipelines, residues that define open states of the barrels are maintained. This minimizes sequence sampling, accelerating the design process. For each of six targets, just two to six synthetic genes are made for expression in Escherichia coli. On average, 70% of these genes express to give soluble monomeric proteins that are fully characterized, including high-resolution structures for most targets that match the design models with high accuracy.

计算蛋白质设计正在迅速发展。在这里，我们描述了从经过验证的平行和反平行肽组装开始，设计两个具有结合小分子的中央通道的α-螺旋桶蛋白家族的有效途径。计算设计以定义的从头寡聚桶状形成肽的序列和结构为种子，并且相邻的螺旋通过环构建连接。对于具有反平行螺旋的目标，短环就足够了。然而，具有平行螺旋的目标需要更长的连接器；即，包装在桶上的螺旋-转角-螺旋-转角-螺旋图案的外层。在整个这些计算管道中，定义桶的打开状态的残基被保留。这最大限度地减少了序列采样，加快了设计过程。对于六个靶标中的每一个，只需制作两到六个合成基因即可在大肠杆菌中表达。平均而言，这些基因中的 70% 表达后可产生完全表征的可溶性单体蛋白，包括大多数靶标的高分辨率结构，与高精度的设计模型相匹配。