Structural and sequence analyses of an infrared fluorescent tissue marker

Abstract

Within the mammalian body infrared (IR) radiation is not scattered by tissues and fluids as much as visible light, making an IR fluorescent marker ideal for imaging applications in mammals. Recently, bacteriophytochromes (Bphs), red‐light sensing proteins from different bacterial strains, have been shown to contain significant levels of IR fluorescence. A Bph from Deinococcus radiodurans, after extensive mutagenesis, has been successfully expressed as IR fluorescent tissue marker in mice. Bph response to light depends on an organic cofactor, biliverdin (BV), which is abundant in the mammalian body. BV, upon the absorption of a photon, induces conformational changes in the surrounding Bph allowing it to reversibly photoconvert between spectrally distinct Pr (red light absorbing) and Pfr (far red light absorbing) states. Bph from Rhodopseudomonas palustris, RpBphP3 (P3), is unique among the Bphs in that its ground state is Pr and its excited state is Pnr (near red light absorbing). P3 and its mutant variants have been shown to emit significant IR fluorescence due to P3's unusual photoconversion properties. A single mutation D216A or Y272F in the BV binding region or C‐terminal truncation of the photosensory module of P3 increases IR fluorescence over the wild‐type protein. Using site‐directed mutagenesis we created mutant variants of P3 that contain combination of D216A or Y272F mutation with C‐terminal truncation of the photosensory module. We report that truncated P3 with D216A mutation displays higher levels of near IR fluorescence relative to wild type P3. Expression and visualization of IR fluorescent P3 mutants in mammalian cell lines followed by in vivo expression in mice are the subsequent goals of this research.Supported by NIH EARDA pilot grant 5G11HD049644 – 04 to E.A.S..