Two-Color Imaging using Spectral Variants of iRFP670 and iRFP682 Near-Infrared Fluorescent Proteins

Abstract

The new class of fluorescent proteins (FPs) engineered from bacterial phytochromes (BphPs) attracts much attention for in vivo imaging due to their near-infrared (NIR) fluorescence spectra. These FPs utilize widely available in mammalian cells biliverdin (BV), a product of heme degradation, as a chromophore and, therefore, are as easy to use as common GFP-like proteins. We recently reported five NIR FPs, called iRFPs, with different fluorescence and biochemical properties. Interestingly, two of them, iRFP670 and iRFP682, exhibited the twice higher molecular brightness, as well as the blue-shifted absorbance (643 nm and 663 nm) and fluorescence (670 nm and 682 nm) compared to other iRFPs. Here we characterized the unusual properties of these NIR FPs in detail. Our biochemical and biophysical analysis showed that iRFP670 and iRFP682 incorporate the BV chromophore in two distinct confirmations. A single amino acid mutation resulted in a depletion of one BV confirmations in the protein binding pocket and, consequently, in 30 nm red-shifts of both absorbance and fluorescence. The point mutation also caused a slight decrease in the molecular brightness and an increase in the pH stability of the obtained red-shifted variants, which we named iRFP670-red and iRFP682-red. The effective brightness of the iRFP670-red and iRFP682-red in live mammalian cells was comparable to that of the parental proteins, suggesting that the high efficiency and high specificity of the incorporation of endogenous BV chromophore was not affected. Spectrally resolvable fluorescence of the iRFP670 and iRFP670-red pair, as well as of the iRFP682 and iRFP682-red pair, allowed easy separation of two cellular populations using FACS cytometry and straightforward two-color fluorescence microscopy of live cells, thus making them the probes of choice for cell labeling in the NIR region.