Effect Of Site-directed Mutagenesis On The Spectroscopic Behavior Of Near-infrared Fluorescent Protein PAiRFP1 And Its Preliminary Research On NIR Biosensor

Bacteriophytochrome(designated as BphP) is one type of BV-containing photosensitive protein widely occurring in various bacteria. It usually exists in the form of Pr(red light-absorbing) and Pfr(far-red light absorbing) state. These two states can be interconverted by using red light or far red light irradiation. Some BphP-based Near-red Fluroesenct Proteins(NIR FPs) have been obtained via protein engineering methods, and then have been successfully applied to NIR fluorescent imaging and NIR photoacustic imaging field in recent years.PAiRFP1 is one NIR FP engineered from Agp2, one bacteriophytochrome Agp2 from Agrobacterium tumefaciens. It has been used as one photoactivable fluorescence probe to detect the tumor in vivo. Little useful information has been available concerning whether PAi RFP1 had other application. In this paper, we explore if PAiRFP1 can used as NIR biosensor specifically for Hg2+. It was found that NIR fluorescence was quenched upon increasing the concentration of Hg2+. Simialr phenomenon was observed for oxidized ions, including Fe3+, Cu2+,Ag+. Unlike theses cases, NIR fluorescene was slightly enhanced upon addition of K+, Mg2+, NH4+, Mn2+ ions mainly due to the photoactivation. These results showed that PAiRFP1 was not suitable for detecting Hg2+.Additionally, PCR site-directed mutagenesis has been also done on V276,C277, C280 residues of PAiRFP1. Spectroscopic data revealed that when compared with other two amino acid residues, V276 residue exerted more significant effect on the maximum absorption position(λmax), the maximum fluorescence emission peak position(Fmax), as well as the NIR fluorescence quantum yield. The mutageneis from Val to Gly caused ~ 15 nm and ~ 11 nm redshift for λmax and Fmax. Meanwhile, the NIR fluorescence quantum yield was decrased to ~ 64.1%. In the next work, we carried out the site-directed saturation mutagenesis on V276 site, and some spectroscopic data have been obtained. These results provided some useful information for the further photoacoustic imaging.