Design of an Infrared Fluorescent Marker for In Vivo Tissue Imaging in Mammals

Infrared fluorescent proteins (IFPs) are ideal for in situ imaging in mammalian tissue because IFPs are less prone to absorbance and light scattering by hemoglobin and water.  Bacteriophytochromes (Bphs) are bacterial red light photoreceptors that have been modified to fluoresce in the infrared (IR) range.  Bphs require an organic cofactor, biliverdin (BV), which is naturally found in the mammalian body, in order to fluoresce.  Recently, a heavily engineered Bph from Deinococcus radiodurans (DrBphP) was successfully expressed in mice as an IR fluorescent tissue marker.  DrBphP exhibits classic Bph photoconversion because it has the ability to photoconvert between red-light and far-red light absorbing forms, Pr and Pfr respectively.  Recently, we found that a Bph from Rhodopseudomonas palustris, RpBphP3 (P3), is naturally fluorescent in the near-IR range.  P3 exhibits unique photoconversion due to the formation an unusual near-red light (Pnr) absorbing form.  A  P3 mutant carrying a single substitution of a highly conserved aspartate-216 to histidine (D216H), introduced in the chromophore-binding pocket has the same fluorescent quantum yield as the heavily engineered DrBphP.  We used site-directed mutagenesis to create a monomeric variant of P3 D216H that will be suited for expression in mammalian cells.  Single mutations were introduced along the P3 dimer interface: threonine-142 to arginine (T142R), glutamate-315 to arginine (E315R), and tryptophan-323 to arginine (W323R).  Our goal is to ultimately express monomeric P3 in mammalian cells and eventually in the mammalian body as IR tissue markers for whole body imaging.