Institut de Biologie Structurale, Université Grenoble Alpes, CEA, CNRS, Grenoble, France.
Institut de Biologie Structurale, Université Grenoble Alpes, CEA, CNRS, Grenoble, France.
Biophys J. 2019 Dec 3;117(11):2087-2100. doi: 10.1016/j.bpj.2019.10.035. Epub 2019 Nov 2.
The availability of fluorescent proteins with distinct phototransformation properties is crucial for a wide range of applications in advanced fluorescence microscopy and biotechnology. To rationally design new variants optimized for specific applications, a detailed understanding of the mechanistic features underlying phototransformation is essential. At present, little is known about the conformational dynamics of fluorescent proteins at physiological temperature and how these dynamics contribute to the observed phototransformation properties. Here, we apply high-resolution NMR spectroscopy in solution combined with in situ sample illumination at different wavelengths to investigate the conformational dynamics of rsFolder, a GFP-derived protein that can be reversibly switched between a green fluorescent state and a nonfluorescent state. Our results add a dynamic view to the static structures obtained by x-ray crystallography. Including a custom-tailored NMR toolbox in fluorescent protein research provides new opportunities for investigating the effect of mutations or changes in the environmental conditions on the conformational dynamics of phototransformable fluorescent proteins and their correlation with the observed photochemical and photophysical properties.
具有独特光转化特性的荧光蛋白的可用性对于高级荧光显微镜和生物技术的广泛应用至关重要。为了合理设计针对特定应用优化的新型变体，必须深入了解光转化背后的机械特征。目前，人们对生理温度下荧光蛋白的构象动力学以及这些动力学如何影响观察到的光转化特性知之甚少。在这里，我们应用高分辨率 NMR 光谱在溶液中结合在不同波长下的原位样品照明来研究 rsFolder 的构象动力学，rsFolder 是一种 GFP 衍生的蛋白质，可以在绿色荧光状态和非荧光状态之间可逆切换。我们的结果为 x 射线晶体学获得的静态结构增加了一个动态视角。在荧光蛋白研究中包含定制的 NMR 工具包为研究突变或环境条件变化对光转化荧光蛋白构象动力学的影响及其与观察到的光化学和光物理性质的相关性提供了新的机会。
