Near infrared fluorescent biliproteins generated from bacteriophytochrome AphB of Nostoc sp. PCC 7120
Near infrared fluorescent biliproteins generated from bacteriophytochrome AphB of Nostoc sp. PCC 7120
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DOI：
10.1039/c5pp00442j
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发表时间：
2016-04
影响因子：
3.1
通讯作者：
Che Yuan-;Haiyang Li;Kun Tang;W. Gärtner;H. Scheer;Ming Zhou;K. Zhao
中科院分区：
文献类型：
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作者：
Che Yuan-;Haiyang Li;Kun Tang;W. Gärtner;H. Scheer;Ming Zhou;K. Zhao
蓝藻念珠菌的基因组。 PCC 7120 编码大量假定的细菌光敏色素和蓝藻色素光感受器，由于它们的长波长吸收和荧光发射，可能作为细胞内研究中的荧光标签。我们发现细菌光敏色素 AphB 的 PAS-GAF 结构域与胆绿素共价结合，除了可逆的光化学作用外，还表现出在近红外 (NIR) 光谱区的中等荧光。选择它是为了进一步提高亮度，同时保留近红外荧光。第一步，被认为可以改善荧光的氨基酸被选择性突变。然后对所得变体进行几轮随机诱变并筛选近红外中增强的荧光。与 wt AphB(1-321) 相比，优化的 PAS-GAF 变体的亮度增加了三倍以上，并且只有微不足道的光谱偏移（A _max 约为 695 nm，F _max 约为 720 nm）。一般来说，亮度随着波长的减小而增加，这允许根据组织的光学特性来选择荧光团。当靠近生色团的 His260 残基突变为 Tyr 时，观察到光谱异质性，这强调了环境对结合的胆绿素生色团的电子特性的强烈影响。
The genome of the cyanobacterium Nostoc sp. PCC 7120 encodes a large number of putative bacteriophytochrome and cyanobacteriochrome photoreceptors that, due to their long-wavelength absorption and fluorescence emission, might serve as fluorescent tags in intracellular investigations. We show that the PAS-GAF domain of the bacteriophytochrome, AphB, binds biliverdin covalently and exhibits, besides its reversible photochemistry, a moderate fluorescence in the near infrared (NIR) spectral region. It was selected for further increasing the brightness while retaining the NIR fluorescence. In the first step, amino acids assumed to improve fluorescence were selectively mutated. The resulting variants were then subjected to several rounds of random mutagenesis and screened for enhanced fluorescence in the NIR. The brightness of optimized PAS-GAF variants increased more than threefold compared to that of wt AphB(1–321), with only insignificant spectral shifts ( A _max around 695 nm, and F _max around 720 nm). In general, the brightness increases with decreasing wavelengths, which allows for a selection of the fluorophore depending on the optical properties of the tissue. A spectral heterogeneity was observed when residue His260, located in close proximity to the chromophore, was mutated to Tyr, emphasizing the strong effects of the environment on the electronic properties of the bound biliverdin chromophore.