Coiled-Coil-Mediated Assembly of an Icosahedral Protein Cage with Extremely High Thermal and Chemical Stability
Coiled-Coil-Mediated Assembly of an Icosahedral Protein Cage with Extremely High Thermal and Chemical Stability
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DOI：
10.1021/jacs.8b13604
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发表时间：
2019-06-12
影响因子：
15
通讯作者：
Marsh, E. Neil G.
中科院分区：
文献类型：
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作者：
Cristie-Dayid, Ajitha S.;Chen, Junjie;Marsh, E. Neil G.
将蛋白质分子组织成高阶纳米级体系结构本质上是无处不在的，它代表了合成生物学的重要目标。此外，酶活性的稳定在生物技术和医学中具有许多实际应用。在这里，我们描述了一种非常稳定的，酶活性的，空心的蛋白笼的对称性设计，该蛋白笼的M-R近似于2.1 MDa，其尺寸与小型二十面体病毒相似。笼子是基于二十面体对称性构建的，该对称性是通过遗传融合的三聚蛋白（Triest）与从头设计的小型五型螺旋线盘绕的线圈结构域构造的，该螺旋螺旋域被柔性寡甘氨酸接头序列隔开。筛选一个小型设计库，其中连接器长度从2到12个残基不等，鉴定出包含8个甘氨酸残基（ICO8）的构造，形成了定义明确的笼子。通过动态光散射，负染色和冷冻EM以及原子力和IR光诱导的力显微镜的表征，ICO8将ICO8组装成一个柔性的空心笼，其中包含20张酯酶三聚体的副本，总共具有60个蛋白质亚基，具有总体icososahedral decosery subinits 。值得注意的是，ICO8形成的笼子被证明在热和化学变性方面非常稳定：而Triest通过加热（t-m大约至75度C）展开Triest，或者由1.5 m甘代盐盐酸盐变性，而ICO8笼子在120摄氏度c cages均处于120摄氏度的折叠。或8 m盐烷盐酸盐。笼子的稳定性提高是一种新特性，它是从蛋白质笼的高阶结构中出现的，而不是固有的，与蛋白质的构成成分是固有的。
The organization of protein molecules into higher order nanoscale architectures is ubiquitous in Nature and represents an important goal in synthetic biology. Furthermore, the stabilization of enzyme activity has many practical applications in biotechnology and medicine. Here we describe the symmetry-directed design of an extremely stable, enzymatically active, hollow protein cage of M-r approximate to 2.1 MDa with dimensions similar to those of a small icosahedral virus. The cage was constructed based on icosahedral symmetry by genetically fusing a trimeric protein (TriEst) to a small pentameric de novo-designed coiled coil domain, separated by a flexible oligo-glycine linker sequence. Screening a small library of designs in which the linker length varied from 2 to 12 residues identified a construct containing 8 glycine residues (Ico8) that formed well-defined cages. Characterization by dynamic light scattering, negative stain, and cryo-EM and by atomic force and IR-photoinduced force microscopy established that Ico8 assembles into a flexible hollow cage comprising 20 copies of the esterase trimer, 60 protein subunits in total, with overall icosahedral geometry. Notably, the cages formed by Ico8 proved to be extremely stable toward thermal and chemical denaturation: whereas TriEst was unfolded by heating (T-m approximate to 75 degrees C) or denatured by 1.5 M guanidine hydrochloride, the Ico8 cages remained folded even at 120 degrees C or in 8 M guanidine hydrochloride. The increased stability of the cages is a new property that emerges from the higher-order structure of the protein cage, rather than being intrinsic to the components from which it is constructed.