European Molecular Biology Laboratory (EMBL), Hamburg Outstation, c/o DESY, Hamburg, Germany.
Biophys J. 2012 Jun 20;102(12):2866-75. doi: 10.1016/j.bpj.2012.05.009. Epub 2012 Jun 19.
Genetically encoded FRET (Foerster resonance energy transfer) sensors are exciting tools in modern cell biology. Changes in the conformation of a sensor lead to an altered emission ratio and provide the means to determine both temporal and spatial changes in target molecules, as well as the activity of enzymes. FRET sensors are widely used to follow phosphorylation events and to monitor the effects of elevated calcium levels. Here, we report for the first time, to our knowledge, on the analysis of the conformational changes involved in sensor function at low resolution using a combination of in vitro and in cellulo FRET measurements and small-angle scattering of x rays (SAXS). The large and dynamic structural rearrangements involved in the modification of the calcium- and phosphorylation-sensitive probe CYNEX4 are comprehensively characterized. It is demonstrated that the synergistic use of SAXS and FRET methods allows one to resolve the ambiguities arising due to the rotation of the sensor molecules and the flexibility of the probe.
遗传编码的 FRET（荧光共振能量转移）传感器是现代细胞生物学中令人兴奋的工具。传感器构象的变化会导致发射比的改变，并提供确定靶分子的时间和空间变化以及酶活性的手段。FRET 传感器广泛用于跟踪磷酸化事件，并监测钙水平升高的影响。在这里，我们首次报道了，据我们所知，使用体外和细胞内 FRET 测量以及 X 射线小角散射（SAXS）的组合，以低分辨率分析传感器功能涉及的构象变化。全面表征了钙和磷酸化敏感探针 CYNEX4 修饰所涉及的大的和动态的结构重排。结果表明，SAXS 和 FRET 方法的协同使用可以解决由于传感器分子的旋转和探针的灵活性而产生的歧义。
