Mutagenesis of mNeptune Red-Shifts Emission Spectrum to 681-685 nm

GFP-like fluorescent proteins with diverse emission wavelengths have been developed through mutagenesis, offering many possible choices in cellular and tissue imaging, such as multi-targets imaging, deep tissue imaging that require longer emission wavelength. Here, we utilized a combined approach of random mutation and structure-based rational design to develop new NIR fluorescent proteins on the basis of a far-red fluorescent protein, mNeptune (Ex/Em: 600/650 nm). We created a number of new monomeric NIR fluorescent proteins with the emission range of 681–685 nm, which exhibit the largest Stocks shifts (77–80 nm) compared to other fluorescent proteins. Among them, mNeptune681 and mNeptune684 exhibit more than 30 nm redshift in emission relative to mNeptune, owing to the major role of the extensive hydrogen-bond network around the chromophore and contributions of individual mutations to the observed redshift. Furthermore, the two variants still maintain monomeric state in solution, which is a trait crucial for their use as protein tags. In conclusion, our results suggest that there is untapped potential for developing fluorescent proteins with desired properties.

通过诱变技术已开发出具备多样发射波长的类绿色荧光蛋白（GFP-like fluorescent proteins），为细胞与组织成像提供了丰富选择，涵盖多靶点成像、需更长发射波长的深层组织成像等应用场景。本研究基于远红色荧光蛋白mNeptune（激发/发射波长：600/650 nm），采用随机诱变与基于结构的理性设计相结合的策略，开发新型近红外（NIR）荧光蛋白。本研究获得了多款新型单体型近红外荧光蛋白，其发射波长范围为681~685 nm，相较其他荧光蛋白展现出最大的斯托克斯位移（Stokes shift，原文作Stocks shift）：77~80 nm。其中，mNeptune681与mNeptune684相较于原始的mNeptune，发射波长产生了超过30 nm的红移，这一现象源于生色团周围广泛的氢键网络所发挥的关键作用，以及各单点突变对观测到的红移的贡献。此外，这两种突变体在溶液中仍保持单体状态，这一特性对于其作为蛋白标签的应用至关重要。综上，本研究结果表明，开发具备理想特性的荧光蛋白仍存在尚未被充分挖掘的潜力。