Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A longstanding design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method that performs rigid three-body fusion of homo-oligomer and spacer building blocks to generate user-defined architectures, while at the same time significantly increasing the number of geometric solutions over typical symmetric fusion. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies from a set of designed homo-dimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from DARPins (designed ankyrin repeat proteins), anchored on one end by α-helical fusion and on the other by a designed homo-dimer interface, and we explored their use for cryo-EM structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects, small scaffold size, and the low-order symmetry of these dihedral scaffolds, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins.

中文翻译：

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使用刚性三体融合生成有序蛋白组装体

蛋白质纳米材料设计是一门新兴学科，在医学及其他领域都有应用。一种长期的设计方法是使用遗传融合通过α-螺旋接头将蛋白质同型低聚物亚基连接起来，以形成更复杂的对称装配，但这种方法因接头的灵活性和缺乏几何解决方案而受阻。在这里，我们描述了一种通用的计算方法，该方法执行均聚物和间隔基团的刚性三体融合以生成用户定义的体系结构，同时与典型的对称融合相比，大大增加了几何解的数量。然后使用Rosetta优化融合结，以最大程度地减少灵活性。我们应用此方法从一组设计的同型二聚体和重复蛋白构建基块设计和测试92个二面对称蛋白装配体。通过天然质谱，小角度X射线散射和负染色单粒子电子显微镜进行的实验验证确认了11种设计的组装状态。这些组件大多数由DARPins（设计为锚蛋白重复蛋白）构建，一端通过α-螺旋融合锚定，另一端通过设计的同二聚体界面锚定，我们通过结合DARPin探索了它们在低温EM结构测定中的用途。选择结合目标靶标的变体。尽管目标分辨率受到首选的定向效应，较小的支架尺寸以及这些二面体支架的低阶对称性的限制，但我们发现双重锚固策略降低了目标DARPIN复合物相对于整体组装的灵活性，