Emory University Department of Chemistry, 1515 Dickey Drive, Atlanta, GA 30322, USA(1); Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Saclay, F-91198 Gif-sur-Yvette CEDEX, France.
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Saclay, F-91198 Gif-sur-Yvette CEDEX, France.
J Struct Biol. 2023 Sep;215(3):108012. doi: 10.1016/j.jsb.2023.108012. Epub 2023 Aug 9.
Synthetic ɑRep repeat proteins are engineered as Brick and Staple protein pairs that together self-assemble into helical filaments. In most cases, the filaments spontaneously form supercrystals. Here, we describe an expanded series of ɑRep Bricks designed to stabilize the interaction between consecutive Bricks, to control the length of the assembled multimers, or to alter the spatial distribution of the Staple on the filaments. The effects of these Brick modifications on the assembly, on the final filament structure and on the crystal symmetry are analyzed by biochemical methods, electron microscopy and small angle X-ray scattering. We further extend the concept of Brick/Staple protein origami by designing a new type of "Janus"-like Brick protein that is equally assembled by orthogonal staples binding its inner or outer surfaces and thus ending inside or outside the filaments. The relative roles of longitudinal and lateral associations in the assembly process are discussed. This set of results demonstrates important proofs-of-principle for engineering these remarkably versatile proteins toward nanometer-to-micron scale constructions.
合成的ɑRep 重复蛋白被设计为 Brick 和 Staple 蛋白对，它们共同自我组装成螺旋丝。在大多数情况下，这些丝自发形成超晶体。在这里，我们描述了一系列扩展的ɑRep Brick，旨在稳定连续 Brick 之间的相互作用，控制组装多聚体的长度，或改变 Staple 在丝上的空间分布。通过生化方法、电子显微镜和小角度 X 射线散射分析这些 Brick 修饰对组装、最终丝结构和晶体对称性的影响。我们通过设计一种新型的“两面神”样 Brick 蛋白进一步扩展 Brick/Staple 蛋白折纸的概念，这种 Brick 蛋白同样可以通过结合其内外表面的正交 Staple 进行组装，从而将其末端置于丝的内部或外部。讨论了在组装过程中纵向和横向缔合的相对作用。这组结果为工程设计这些非常通用的蛋白质提供了重要的原理证明，朝着纳米到微米级结构的方向发展。
