Rational Structure-Based Design of Bright GFP-Based Complexes with Tunable Dimerization
Rational Structure-Based Design of Bright GFP-Based Complexes with Tunable Dimerization
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具有可调二聚化作用的基于 GFP 的明亮复合物的合理结构设计
DOI：
10.1002/anie.201506686
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发表时间：
2015-11-16
影响因子：
16.6
通讯作者：
Chen, Swaine L.
中科院分区：
文献类型：
--
作者：
Eshaghi, Majid;Sun, Guangyu;Chen, Swaine L.
荧光蛋白是变革性的工具;因此，任何亮度的增加都是受欢迎的改进。我们发明了“vGFP策略”，该策略基于GFP与单结构域抗体结合的结构分析，预测可调的二聚化，增强的亮度（约100 μ m），50%），并改善pH值的阻力。我们使用生物化学，晶体学和单分子研究验证了所有这些预测。我们在三种不同的情况下应用vsfGFP蛋白：体外单步免疫荧光（由于二聚化，亮度提高了3倍）;体内细菌和人类细胞中的表达（亮度提高了1.5倍）;体内人类细胞中显示出更好的pH抗性的蛋白融合。因此，vGFP策略允许现有应用的升级，适用于其他荧光蛋白，并建议用于调节任意蛋白质的二聚化和优化蛋白质性质的方法。
Fluorescent proteins are transformative tools; thus, any brightness increase is a welcome improvement. We invented the "vGFP strategy" based on structural analysis of GFP bound to a single-domain antibody, predicting tunable dimerization, enhanced brightness (ca. 50%), and improved pH resistance. We verified all of these predictions using biochemistry, crystallography, and single-molecule studies. We applied the vsfGFP proteins in three diverse scenarios: single-step immunofluorescence in vitro (3 x brighter due to dimerization); expression in bacteria and human cells in vivo (1.5 x brighter); and protein fusions showing better pH resistance in human cells in vivo. The vGFP strategy thus allows upgrading of existing applications, is applicable to other fluorescent proteins, and suggests a method for tuning dimerization of arbitrary proteins and optimizing protein properties in general.