Identifying Novel Fluorescent Bio-Markers

Bacteriophytochromes (Bphs) are bacterial red-light photoreceptors that have been recently engineered for use as infrared (IR) fluorescent tissue markers. Bphs are ideal proteins for for in vivo tissue imaging because IR signals are less prone to absorption and light scattering by hemoglobin and water. In addition, Bph fluorescence relies on a covalently bound co-factor, biliverdin (BV), which is naturally abundant in mammalian tissue. Upon absorption of a photon, BV undergoes extensive structural changes, affecting the surrounding amino acids and ultimately the protein. Bph from Rhodopseudomonas palustris, RpBphP3 (P3) goes through restricted photoconversion and therefore is naturally more fluorescent than other Bphs. Exploring photoreceptors homologous to P3 is imperative in identifying bio-markers with IR fluorescent properties. Using BLAST (Basic Local Alignment Search Tool) we identified a putative Bph from Stigmatella aurantiaca, a gram-negative myxobacterim that forms fruiting bodies in nutrient deprived conditions. Photoreceptors such as Bph may be used by soil-dwelling S. aurantiaca to gather geo-directional information to form fruiting bodies closer to the soil surface.  Interestingly, SaBphP contains amino acid threonine (Thr) in a key position that stabilizes BV in the Pr state instead of a highly conserved histidine (His).  We successfully cloned and expressed the gene coding for SaBphP in E. coli. The purified SaBphP was analyzed using UV-visible absorption and fluorescence spectrophotometry. SaBphP, similar to P3, undergoes limited photoconversion and is naturally fluorescent in the IR range. Utilizing site-directed mutagenesis we are planning to optimize SaBphP for potential use as IR tissue marker.