光激活近红外荧光蛋白PAiRFP1的多色成像改造初步研究

Modification of Photoactivatable Near-Infrared Fluorescent Protein PAiRFP1 for Multicolor Imaging

遗传学编码的光激活近红外荧光蛋白PAiRFP1是近年来活体成像领域中的一个非常有潜力的荧光标记蛋白,发射峰在720 nm左右,可有效提高成像的信噪比。为了实现多色成像,采用PCR定点突变,将GAF结构域中第247位异亮基酸(Ile)突变为半胱氨酸(Cys),该Cys残基在其他相关荧光蛋白中对于光谱的蓝移发挥了重要作用。通过蛋白质工程、体外BV组装、以及光谱学检测发现:该Cys残基的引入对于PAiRFP1近红外荧光发射最大峰位置并未产生显著影响,但却降低了摩尔消光系数、荧光量子产率等参数。Pymol分析显示:光谱未发生蓝移的原因可能是因为Cys无法与BV中A环中的特定的双键形成硫醚键所致,相光工作尚待深入研究。

The photoactivatable near-infrared fluorescent protein PAiRFP1 is an promising,achieving higher SNR NIR fluorescent protein emitting at 720 nm.In order to achieve multicolor imaging,here we utilized PCR site-directed mutagenesis to substitute isoleucine(Ile)at position 247 in the GAF domain to cysteine(Cys).Spectroscopic data showed that unlike other BphP,there was no big spectral blue shift for the maximum fluorescence emission peak.Nevertheless,the molar extinction coefficient and fluorescence quantum yield of the PAiRFP1/I247 C mutant were significantly reduced.Pymol analysis was done for gaining some explanation.