Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, United States.
Department of Chemistry, State University of New York, Buffalo, New York 14260, United States.
Biochemistry. 2023 Feb 7;62(3):770-781. doi: 10.1021/acs.biochem.2c00595. Epub 2023 Jan 12.
The design of functional proteins requires specification of tertiary structure and incorporation of molecular binding sites. Here, we develop an inside-out design strategy in the molecular modeling program Rosetta that begins with amino acid side chains from one or two α-helices making well-defined contacts with a ligand. A full-sized protein is then built around the ligand by adding additional helices that promote the formation of a protein core and allow additional contacts with the ligand. The protocol was tested by designing 12 zinc-binding proteins, each with 4-5 helices. Four of the designs were folded and bound to zinc with equilibrium dissociation constants varying between 95 nM and 1.1 μM. The design with the tightest affinity for zinc, N12, adopts a unique conformation in the folded state as assessed with nuclear magnetic resonance (NMR) and the design model closely matches (backbone root-mean-square deviation (RMSD) < 1 Å) an AlphaFold model of the sequence. Retrospective analysis with AlphaFold suggests that the sequences of many of the failed designs did not encode the desired tertiary packing.
功能蛋白的设计需要指定三级结构并纳入分子结合位点。在这里，我们在分子建模程序 Rosetta 中开发了一种从内到外的设计策略，该策略从一个或两个α-螺旋的氨基酸侧链开始，与配体形成明确的接触。然后，通过添加促进形成蛋白质核心并允许与配体进一步接触的额外螺旋，在配体周围构建全尺寸蛋白质。该方案通过设计 12 个锌结合蛋白进行了测试，每个蛋白有 4-5 个螺旋。其中 4 个设计折叠并与锌结合，平衡解离常数在 95 nM 和 1.1 μM 之间变化。与锌结合亲和力最强的设计 N12 在折叠状态下采用独特的构象，如通过核磁共振 (NMR) 评估和设计模型与序列的 AlphaFold 模型非常匹配（骨架均方根偏差 (RMSD) < 1 Å）。使用 AlphaFold 进行的回顾性分析表明，许多失败设计的序列没有编码所需的三级包装。
