Lerma Romero Jorge A, Kolmar Harald
Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, Germany.
Centre for Synthetic Biology, Technical University of Darmstadt, Darmstadt, Germany.
Methods Mol Biol. 2023;2681:249-274. doi: 10.1007/978-1-0716-3279-6_14.
The binding pocket of some therapeutic targets can acquire multiple conformations that, to some extent, depend on the protein dynamics and the interaction with other molecules. The inability to reach the binding pocket can impose a substantial or even insurmountable barrier for the de novo identification or optimization of small-molecule ligands. Herein, we describe a protocol for the engineering of a target protein and a yeast display FACS sorting strategy to identify protein variants with a stable transient binding pocket with improved binding for a cryptic site-specific ligand. This strategy may facilitate drug discovery using the resulting protein variants with accessible binding pockets for ligand screening.
一些治疗靶点的结合口袋可以获得多种构象，这些构象在一定程度上取决于蛋白质动力学以及与其他分子的相互作用。无法到达结合口袋可能会对小分子配体的从头鉴定或优化造成重大甚至难以克服的障碍。在此，我们描述了一种工程改造目标蛋白的方案以及一种酵母展示荧光激活细胞分选策略，以鉴定具有稳定瞬时结合口袋且对隐蔽位点特异性配体结合能力增强的蛋白变体。这种策略可能会促进利用所得具有可及结合口袋的蛋白变体进行配体筛选的药物发现。
