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首页> 外文期刊>Biochemistry >Intrinsic Dynamics in ECFP and Cerulean Control Fluorescence Quantum Yield
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Intrinsic Dynamics in ECFP and Cerulean Control Fluorescence Quantum Yield

机译：ECFP和Cerulean控制荧光量子产率的内在动力学

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Enhanced cyan fluorescent protein (ECFP) and its variant Cerulean are genetically encoded fluorophores widely used as donors in FRET-based cell imaging experiments. First, we have confirmed through denaturation experiments that the double-peak spectroscopic signature of these fluorescent proteins originates from the indole ring of the chromophore. Then, to explain the improvement in the fluorescence properties of Cerulean compared to those of ECFP, we have determined the high-resolution crystal structures of these two proteins at physiological pH and performed molecular dynamics simulations. In both proteins, the N-terminal half of the seventh strand exhibits two conformations. These conformations both have a complex set of van der Waals interactions with the chromophore and, as our simulations suggest, they interconvert on a nanosecond time scale. The Y145A and H148D mutations in Cerulean stabilize these interactions and allow the chromophore to be more planar, better packed, and less prone to collisional quenching, albeit only intermittently. As a consequence, the probability of nonradiative decay is significantly decreased. Our results highlight the considerable dynamical flexibility that exists in the vicinity of the tryptophan-based chromophore of these engineered fluorescent proteins and provide insights that should allow the design of mutants with enhanced optical properties.
机译：增强型青色荧光蛋白（ECFP）及其变体Cerulean是遗传编码的荧光团，广泛用作基于FRET的细胞成像实验中的供体。首先，我们已经通过变性实验证实了这些荧光蛋白的双峰光谱特征来自发色团的吲哚环。然后，为了说明与ECFP相比Cerulean的荧光性质有所改善，我们确定了这两种蛋白质在生理pH下的高分辨率晶体结构，并进行了分子动力学模拟。在这两种蛋白质中，第七条链的N端一半都显示两个构象。这些构象都与发色团具有一组复杂的范德华相互作用，并且正如我们的模拟所暗示的，它们在纳秒级的时间尺度上相互转换。 Cerulean中的Y145A和H148D突变稳定了这些相互作用，并使发色团更平整，堆积更好，并且不容易发生碰撞猝灭，尽管只是间歇性的。结果，非辐射衰减的可能性大大降低。我们的结果突出了这些工程化荧光蛋白基于色氨酸的发色团附近存在的相当大的动力学灵活性，并提供了可用于设计具有增强光学特性的突变体的见识。

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