Choi Hyeokjune, Park Hyoin, Son Kabi, Kim Ho Min, Jung Yongwon
Department of Chemistry , KAIST , 291 Daehak-ro, Yuseong-gu , Daejeon 34143 , Republic of Korea . Email:
Graduate School of Medical Science & Engineering , KAIST , Daejeon 34141 , Republic of Korea.
Chem Sci. 2019 Oct 7;10(44):10428-10435. doi: 10.1039/c9sc04158c. eCollection 2019 Nov 28.
Supramolecular protein assemblies have garnered considerable interest due to their potential in diverse fields with unrivaled attainable functionalities and structural accuracy. Despite significant advances in protein assembly strategies, inserting long linkers with varied lengths and rigidity between assembling protein building blocks remains extremely difficult. Here we report a series of green fluorescent protein (GFP) oligomers, where protein building blocks were linked two independent peptide strands. Assembling protein units for this two-peptide assembly were designed by flopped fusion of three self-assembling GFP fragments with two peptide linkers. Diverse flexible and rigid peptide linkers were successfully inserted into high-valent GFP oligomers. In addition, oligomers with one flexible linker and one rigid linker could also be fabricated, allowing more versatile linker rigidity control. Linker length could be varied from 10 amino acids (aa) even up to 76 aa, which is the longest among reported protein assembling peptide linkers. Discrete GFP oligomers containing diverse linkers with valencies between monomers to decamers were monodispersely purified by gel elution. Furthermore, various functional proteins could be multivalently fused to the present GFP oligomers. Binding assays, size exclusion chromatography, dynamic light scattering, circular dichroism, differential scanning calorimetry, and transmission electron microscopy suggested circular geometries of the GFP oligomers and showed distinct characteristics of GFP oligomers with length/rigidity varied linkers. Lastly, a surface binding study indicated that more spaced oligomeric binding modules offered more effective multivalent interactions than less spaced modules.
超分子蛋白质组装体因其在不同领域具有潜在的功能和无与伦比的结构准确性而备受关注。尽管蛋白质组装策略取得了重大进展，但在组装蛋白质构建块之间插入具有不同长度和刚性的长连接子仍然极其困难。在此，我们报道了一系列绿色荧光蛋白（GFP）寡聚体，其中蛋白质构建块由两条独立的肽链连接。这种双肽组装的蛋白质组装单元是通过将三个自组装的GFP片段与两个肽连接子进行翻转融合设计而成。各种柔性和刚性肽连接子成功插入到高价GFP寡聚体中。此外，还可以制备具有一个柔性连接子和一个刚性连接子的寡聚体，从而实现更灵活的连接子刚性控制。连接子长度可以从10个氨基酸（aa）变化到76个aa，这是报道的蛋白质组装肽连接子中最长的。通过凝胶洗脱法对含有不同连接子、单体至十聚体价态的离散GFP寡聚体进行了单分散纯化。此外，各种功能蛋白可以多价融合到目前的GFP寡聚体上。结合测定、尺寸排阻色谱、动态光散射、圆二色性、差示扫描量热法和透射电子显微镜表明GFP寡聚体具有圆形几何结构，并显示出连接子长度/刚性不同的GFP寡聚体的独特特征。最后，一项表面结合研究表明，间隔更大的寡聚结合模块比间隔较小的模块提供更有效的多价相互作用。
