Abstract

Bright monomeric near-infrared fluorescent proteins (NIR-FPs) are useful as markers for labeling proteins and cells and as sensors for reporting molecular activities in living cells and organisms. However, current monomeric NIR-FPs are dim under excitation with common 633/635/640 nm lasers, limiting their broad use in cellular/subcellular level imaging. Here, we report a bright monomeric NIR-FP with maximum excitation at 633 nm, named mIFP663, engineered from Xanthomonas campestris pv Campestris phytochrome (XccBphP). mIFP663 has high molecular brightness with a large extinction coefficient (86,600 M-1 cm-1) and a decent quantum yield (19.4%), and high cellular brightness that is 3-6 times greater than those of spectrally similar NIR-FPs in HEK293T cells in the presence of exogenous BV. Moreover, we demonstrate that mIFP663 is able to label critical cellular and viral proteins without perturbing subcellular localization and virus replication, respectively. Finally, with mIFP663, we engineer improved bimolecular fluorescence complementation (BiFC) and new bioluminescent resonance energy transfer (BRET) systems to detect protein-protein interactions in living cells.