Robust De Novo-Designed Homotetrameric Coiled Coils

四聚体 螺旋线圈 蛋白质设计 合成生物学 蛋白质工程 稳健性(进化) 结构生物学 蛋白质稳定性 蛋白质结构 结构母题 三聚体 蛋白质折叠 生物物理学 化学 计算生物学 生物化学 生物 二聚体 有机化学 基因
作者
Caitlin L. Edgell,Nigel J. Savery,Derek N. Woolfson
出处
期刊:Biochemistry [American Chemical Society]
卷期号:59 (10): 1087-1092 被引量:11
标识
DOI:10.1021/acs.biochem.0c00082
摘要

De novo-designed protein domains are increasingly being applied in biotechnology, cell biology, and synthetic biology. Therefore, it is imperative that these proteins be robust to superficial changes; i.e., small changes to their amino acid sequences should not cause gross structural changes. In turn, this allows properties such as stability and solubility to be tuned without affecting structural attributes like tertiary fold and quaternary interactions. Reliably designed proteins with predictable behaviors may then be used as scaffolds to incorporate function, e.g., through the introduction of features for small-molecule, metal, or macromolecular binding, and enzyme-like active sites. Generally, achieving this requires the starting protein fold to be well understood. Herein, we focus on designing α-helical coiled coils, which are well studied, widespread, and often direct protein–protein interactions in natural systems. Our initial investigations reveal that a previously designed parallel, homotetrameric coiled coil, CC-Tet, is not robust to sequence changes that were anticipated to maintain its structure. Instead, the alterations switch the oligomeric state from tetramer to trimer. To improve the robustness of designed homotetramers, additional sequences based on CC-Tet were produced and characterized in solution and by X-ray crystallography. Of these updated sequences, one is robust to truncation and to changes in surface electrostatics; we call this CC-Tet*. Variants of the general CC-Tet* design provide a set of homotetrameric coiled coils with unfolding temperatures in the range from 40 to >95 °C. We anticipate that these will be of use in applications requiring robust and well-defined tetramerization domains.
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