Transmembrane β-barrel proteins (TMBs) are of great interest for single-molecule analytical technologies because they can spontaneously fold and insert into membranes and form stable pores, but the range of pore properties that can be achieved by repurposing natural TMBs is limited. We leverage the power of de novo computational design coupled with a “hypothesis, design, and test” approach to determine TMB design principles, notably, the importance of negative design to slow β-sheet assembly. We design new eight-stranded TMBs, with no homology to known TMBs, that insert and fold reversibly into synthetic lipid membranes and have nuclear magnetic resonance and x-ray crystal structures very similar to the computational models. These advances should enable the custom design of pores for a wide range of applications.

跨膜 β-桶蛋白 (TMB) 对单分子分析技术非常感兴趣，因为它们可以自发折叠并插入膜中并形成稳定的孔，但通过重新利用天然 TMB 可以实现的孔特性范围有限。我们利用从头计算设计的力量以及“假设、设计和测试”方法来确定 TMB 设计原则，特别是负设计对减慢 β-折叠组装的重要性。我们设计了新的八链 TMB，与已知的 TMB 没有同源性，它们可逆地插入并折叠到合成脂质膜中，并且具有与计算模型非常相似的核磁共振和 X 射线晶体结构。这些进步应该能够为广泛的应用定制孔设计。