Structure‐based engineering of an infrared fluorescent protein marker

Abstract

Infrared fluorescent proteins (IFPs) emit a signal less prone to absorbance by hemoglobin and water, making them ideal candidates for mammalian imaging. Bacteriophytochromes (Bphs), bacterial red‐light photoreceptors, are an optimal source of IFPs. The required cofactor for Bph photochemistry, biliverdin (BV), exists naturally in mammalian cells as a product of heme metabolism. Recently, RpBphP2 (P2) from Rhodopseudomonas palustris was successfully engineered as an IFP marker for noninvasive mammalian imaging. RpBphP3 (P3), which shares a 52% sequence identity with P2, is naturally fluorescent due to an unusually long excited‐state lifetime. Comparative sequence and structural analyses of Bphs identified a highly conserved Asp residue essential for stabilization of BV in the chromophore‐binding domain. Using site‐directed mutagenesis we introduced a single amino acid substitution, D216H, effectively disrupting the photochemistry of P3 as observed by UV‐vis absorption spectroscopy. In addition, the P3 D216H mutant displays a higher fluorescence quantum yield relative to wild type P3. We cloned P3 D216H into the mammalian expression vector, Adtrack‐CMV. Stability of this protein marker will be characterized using in vitro comparison studies of protein maturation with the P2‐derived IFP. Subsequent research goals include expression and visualization of the P3 mutant in HEK293 and HeLa cell lines.