Abstract
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.
Highlights
Fluorescent proteins (FPs) are widely used as protein tags for cellular and in vivo molecular targeting as well as tracking
A number of NIR FPs have been designed through mutagenesis, such as eqFP670 [3] and TagRFP675 [4], reaching maximum emission wavelengths of 670 nm and above
In order to screen for red-shifted proteins, we started with mutagenesis using random mutation of the entire protein
Summary
Fluorescent proteins (FPs) are widely used as protein tags for cellular and in vivo molecular targeting as well as tracking. A number of NIR FPs have been designed through mutagenesis, such as eqFP670 [3] and TagRFP675 [4], reaching maximum emission wavelengths of 670 nm and above. Monomeric NIR variants, rather than other forms, such as dimers or tetramers, are important for labeling interest proteins in vivo in order to avoid the labeled proteins aggregation caused by FP oligomerization characteristics. There are still lack of such NIR proteins. At the beginning of our study, a bright monomeric autocatalytic FP derived from eqFP578 [5,6] of the sea anemone Entacmaea quadricolor, mNeptune [7]
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