Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering.
Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering.
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DOI：
10.1002/cbic.202200321
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发表时间：
2022-08-17
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--
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纳米抗体作为体内操纵和可视化蛋白质的工具越来越受欢迎。 使用光控制纳米抗体/抗原相互作用的能力可以提供对蛋白质功能的精确时空控制。我们开发了一种通用方法来使用通过遗传密码扩展引入靶结合界面的光笼氨基酸来工程化可光活化的纳米抗体。在计算丙氨酸扫描和分子动力学模拟的指导下，我们调整纳米抗体/靶结合亲和力，以在解开之前消除结合。在使用365 nm光的光活化后，结合恢复。 我们使用这种方法来产生两种抗GFP纳米抗体的改进的光笼化变体，当直接在复杂的细胞内环境中与其抗原一起表达时，这些变体功能强大。我们应用它们来控制线虫秀丽隐杆线虫的亚细胞蛋白定位。我们的方法直接在活体动物中应用来自计算建模的预测，并证明了解释体内蛋白质相互作用的重要性。 使用遗传密码扩展和结合界面的计算引导工程的组合来创建用于细胞内使用的光活化纳米抗体。工程化的纳米抗体允许在活体秀丽隐杆线虫中对亚细胞蛋白定位进行光学控制。
Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo‐activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo‐activation using 365 nm light, binding is restored. We use this approach to generate improved photocaged variants of two anti‐GFP nanobodies that function robustly when directly expressed in a complex intracellular environment together with their antigen. We apply them to control subcellular protein localisation in the nematode worm Caenorhabditis elegans. Our approach applies predictions derived from computational modelling directly in a living animal and demonstrates the importance of accounting for in vivo effects on protein‐protein interactions. Light‐activated nanobodies for intracellular use were created using a combination of genetic code expansion and computationally guided engineering of the binding interface. The engineered nanobodies allow optical control of subcellular protein localisation in living Caenorhabditis elegans.
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