Structure-based engineering of an infrared fluorescent protein marker

Infrared fluorescent proteins (IFPs) are optimal for in vivo cell imaging because the infrared (IR) signal is less prone to absorbance by hemoglobin, melanin, and water. Bacteriophytochromes (Bphs) are bacterial red-light photoreceptors that have been successfully engineered as IFPs, and used for imaging of internal organs such as the liver. Bphs require an organic cofactor, biliverdin (BV) for their photochemistry. BV, a linear tetrapyrrole and derivative of heme metabolism, naturally occurs in mammalian tissues. Recently, a Bph from Rhodopseudomonas palustris RpBphP2 (P2) was engineered as a successful IFP marker using a variety of mutations. P2 in combination with related RpBphP3 (P3) modulates synthesis of a light-harvesting complex in R. palustris. Although P2 and P3 share 52% amino acid sequence identity, they have distinct photochemical properties.  Classical Bphs such as P2 undergo classical photoconversion between red and far-red light absorbing states, denoted Pr and Pfr respectively. In addition, they have low fluorescence quantum yield. P3 is naturally fluorescent because of an unusually long excited state lifetime, which translates into a significantly increased fluorescence quantum yield as compared to classical Bphs. P3 is unique because it undergoes photoconversion between Pr and near-red light absorbing state, denoted Pnr. Using site-directed mutagenesis, we are introducing single amino acid substitutions in the BV-binding pocket of P3. We will characterize each of the mutants using UV-vis absorption and fluorescence spectroscopy with the goal of engineering a bright and stable IFP that can be used to image deep tissue in the mammalian body.