Yang Shudan, Jin Shikai, Zhang Mengxi, Chen Yuda, Guo Yiming, Hu Yu, Wolynes Peter G, Xiao Han
Department of Chemistry, Rice University, Houston, TX, USA.
Center for Theoretical Biological Physics, Rice University, Houston, TX, USA.
Nat Chem Biol. 2025 Sep 11. doi: 10.1038/s41589-025-02003-1.
Fluorescent protein fusions with environmentally sensitive fluorophores have been widely used to investigate changes in the protein microenvironment. Unfortunately, these techniques often rely on bulky fluorescent proteins or tags to the N terminus or C terminus of the target protein, which can disrupt the behavior of the target protein and may limit their ability to investigate microenvironment changes with high spatial resolution. Here we develop a strategy to visualize microenvironment changes of protein substructures in real time by genetically incorporating environment-sensitive noncanonical amino acids (ncAAs) containing rotor-based fluorophores at specific positions of the target protein. Through computational redesign of aminoacyl-tRNA synthetase, we successfully incorporated these rotor-based ncAAs into several proteins in mammalian cells. Precise placement of these ncAAs at specific sites of proteins enables the detection of microenvironmental changes around individual residues during events such as aggregation, clustering, cluster dissociation and others.
与环境敏感荧光团融合的荧光蛋白已被广泛用于研究蛋白质微环境的变化。不幸的是，这些技术通常依赖于庞大的荧光蛋白或与靶蛋白N端或C端的标签，这可能会破坏靶蛋白的行为，并可能限制其以高空间分辨率研究微环境变化的能力。在这里，我们开发了一种策略，通过在靶蛋白的特定位置基因掺入含基于转子的荧光团的环境敏感非标准氨基酸（ncAAs），实时可视化蛋白质亚结构的微环境变化。通过对氨酰-tRNA合成酶的计算重新设计，我们成功地将这些基于转子的ncAAs掺入哺乳动物细胞中的几种蛋白质中。将这些ncAAs精确放置在蛋白质的特定位点，能够在诸如聚集、聚类、簇解离等事件期间检测单个残基周围的微环境变化。
