School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan.
ACS Synth Biol. 2022 Jun 17;11(6):2144-2153. doi: 10.1021/acssynbio.2c00058. Epub 2022 Mar 18.
Protein assemblies forming nano- to micro-sized structures underlie versatile biological events in living systems. For mimicking and engineering these protein assemblies through a bottom-up approach, self-assembling peptides (SAPs) that form nanofibril structures via β-sheets serve as potential practical tags. Nevertheless, the development of SAP tags is still in its infancy, and insight into the relationship between peptide sequences and intracellular self-assembly is limited. In this study, we focused on hydrophobic residues in SAPs and examined the self-assembly of SAP-tagged superfolder GFPs (green fluorescent proteins) in COS-7 cells. Based on XEXK (X; hydrophobic amino acids: F, L, I, V, W, or Y) sequence units, we designed a panel of X peptides with different hydrophobic residues (X) and chain lengths (). We observed that the self-assembly propensity, the size of the assemblies, the influence on protein denaturation, and the subcellular localization differed significantly depending on the hydrophobic amino acid. F9, L9, I7, and V13 peptides formed μm-scaled granules, W13 formed small oligomeric clusters in the cytoplasm, and Y15 formed assemblies in the nucleus. In addition, we investigated the orthogonality of their interaction. Strikingly, W13- and Y15-tagged proteins interacted independently and formed two distinct assemblies in cells. Herein, we have demonstrated the great opportunities for rationalizing artificial protein assemblies and orthogonal structures in an intracellular context using the designed SAPs.
蛋白质组装体形成纳米到微米大小的结构，为生命系统中的多种生物学事件提供基础。为了通过自下而上的方法模拟和设计这些蛋白质组装体，通过β-折叠形成纳米纤维结构的自组装肽（SAP）可以作为潜在的实际标签。然而，SAP 标签的开发仍处于起步阶段，对肽序列与细胞内自组装之间关系的了解有限。在这项研究中，我们专注于 SAP 中的疏水性残基，并研究了 SAP 标记的超折叠 GFP（绿色荧光蛋白）在 COS-7 细胞中的自组装。基于 XEXK（X；疏水性氨基酸：F、L、I、V、W 或 Y）序列单元，我们设计了一组具有不同疏水性残基（X）和链长（）的 X 肽。我们观察到，自组装倾向、组装体的大小、对蛋白质变性的影响以及亚细胞定位因疏水性氨基酸而异。F9、L9、I7 和 V13 肽形成了 μm 级的颗粒，W13 在细胞质中形成了小的寡聚体簇，Y15 在核中形成了组装体。此外，我们研究了它们相互作用的正交性。引人注目的是，W13 和 Y15 标记的蛋白质独立相互作用，并在细胞中形成两个不同的组装体。在此，我们使用设计的 SAP 证明了在细胞内环境中合理构建人工蛋白质组装体和正交结构的巨大机会。
