Molecular Basis of Spectral Diversity in Near-Infrared Phytochrome-Based Fluorescent Proteins.
Molecular Basis of Spectral Diversity in Near-Infrared Phytochrome-Based Fluorescent Proteins.
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DOI：
10.1016/j.chembiol.2015.10.007
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发表时间：
2015-11-19
影响因子：
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通讯作者：
Verkhusha VV
中科院分区：
文献类型：
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作者：
Shcherbakova DM;Baloban M;Pletnev S;Malashkevich VN;Xiao H;Dauter Z;Verkhusha VV
由细菌光敏色素（BphPs）制成的近红外荧光蛋白（NIR FPs）是深层组织成像的首选探针。几个过程的检测需要光谱上不同的近红外FPs。我们开发了一种近红外FP， BphP1-FP，它在bphp1衍生的FP中具有最多的蓝移光谱和最高的荧光量子产率。我们发现这些特性是由于胆绿素发色团与GAF结构域的半胱氨酸残基结合而产生的，这与天然BphPs和其他基于bphp的FPs不同。为了阐明谱移的分子基础，我们应用生化、结构和质谱分析揭示了独特发色团的形成。不同来源的近红外FPs的突变表明，光谱位移的机制是普遍的，可用于从其他BphPs设计多色近红外FPs。我们将对光谱上不同的点半胱氨酸突变体应用于多色细胞标记，并证明它们在模型深层组织成像中表现良好。
Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are the probes of choice for deep-tissue imaging. Detection of several processes requires spectrally distinct NIR FPs. We developed an NIR FP, BphP1-FP, which has the most blue-shifted spectra and the highest fluorescence quantum yield among BphP-derived FPs. We found that these properties result from the binding of the biliverdin chromophore to a cysteine residue in the GAF domain, unlike natural BphPs and other BphP-based FPs. To elucidate the molecular basis of the spectral shift, we applied biochemical, structural and mass spectrometry analyses and revealed the formation of unique chromophore species. Mutagenesis of NIR FPs of different origins indicated that the mechanism of the spectral shift is general and can be used to design multicolor NIR FPs from other BphPs. We applied pairs of spectrally distinct point cysteine mutants to multicolor cell labeling and demonstrated that they perform well in model deep-tissue imaging.