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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.

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