Functional and Structural Characterization of A New Monomeric Far-Red Fluorescent Protein

Abstract

Fluorescent proteins have become valuable tools for biomedical research as protein tags, reporters of gene expression, biosensor components, and cell lineage tracers. However, applications of fluorescent proteins for deep tissue imaging have been constrained by the opacity of tissues to excitation light below 600 nm, due to absorbance by hemoglobin. Fluorescent proteins that excite efficiently in the “optical window” above 600 nm are therefore highly desirable. We report here the evolution of a far-red fluorescent protein with peak excitation at 600 nm and peak emission at 650 nm. This, Neptune, performs well in imaging deep tissues in living mice. The crystal structure of Neptune reveals novel mechanisms for red-shifting, including the acquisition of a new hydrogen bond with the chromophore. Neptune may serve as the basis for fluorescent indicators or FRET reporters that are more compatible with deep tissue imaging.