Study Of Screening Near-infrared Fluorescence Probes From Cyanobacterial Phytochrome And Phycobiliprotein

There are lots of pigment-proteins can capture light energy in Cyanobacteria,they are similar to plant phytochrome and bacteriophytochrome,they also have far red light and red light regulation characteristics,so we called them cyanobacteriochrome.Cyanobacteriochrome AphB from Nostoc sp.PCC 7120 can capture light energy,it has PAS and GAF domain,the 17 Cys of PAS domain covalent bind the C3 atoms of A ring of biliverdin molecules,and the GAF domain wrap the pigment molecules tightly inside the protein through the space structure.Because of the existence of biliverdin,pigment-protein has a characteristic spectrum,and the absorption peak of pigment-protein is at 697 nm,the fluorescence peak of pigment-protein is at 720 nm,which locates in visualization in living tissues in vivo imaging window(650-1100 nm),thus it is a great potential marerial for near-infrared fluorescent probes.In this article,according to the conservative amino acid sites which were found in some bacteriophytochromes,we evolved some mutunts of AphB mainly through site-directed mutagenesis and random mutagenesis of conservative amino acid sites.We aim to evolve the better mutant which has good characters of stability in order to develop fluorescent probes in vivo.First according to the study of AphB in our lab,we found that AphB(7-321/A288V)has better light stability than Wild type,and then we construct mutation Asp207 into Ala207,Gly207,Thr207,and Phe207 respectively to study the function of Asp207.O n the basis of these mutations,we do random mutation of protein through error-prone PCR and DN A shuffling,construct mutant library and screening by a high flux instrument Qpix 420,then we select mutant with multifunctional enzyme mark and fluorescence spectrometer,then we do protein expression in E.coli,purification,spectrum detection,analyze physical and chemical properties of mutant.We compare the sequence of mutant through T-coffee O nline to analyze amino acid sites.RpBphP2(PDB: 4e04)as the template,we construct three-dimensional model of PAS-GAF of AphB by the online homology modeling SWISS-MODEL software.Among the nine mutations,the absorption and fluorescence peak position has 2 nm blue shift,stokes shift are 24 nm,the best fluorescent quantity yield increase by 160%,the best extinction coefficient increase by 130%,the best relative molecular brightness increased by 200%,when compared with template.O ur work about AphB can provide an advice for near-infrared fluorescent probe of cyanobacterial phytochrome.In addition,studies have shown that,phycobiliprotein named smURFP can bind BV which are sufficient in mammalian cell,and it has stable and powerful fluorescence that are important for probes.ApcE from Nostoc sp.PCC 7120 is the core-membrane linker protein of phycobilisome which can combine with phycobilin PCB or PEB to form homologous protein.It can not bind biliverdin so we want to screen a mutunt can bind BV for the near-infrared fluorescent probes.According to the study of Apc E from Nostoc sp.PCC 7120 in our laboratory,first we do site-directed mutations to get a better mutant of ApcE,then we take the mutant as template do error-prone PC R to construct ApcE(50-240/(35)77-153)combined with BV homologous protein mutant library.We use high flux instrument Qpix 420 for screening,then we do protein expression in E.coli,purification,spectrum detection,analyze physical and chemical properties of mutant.We compare the sequence of mutant through T-coffee O nline to analyze amino acid sites.As a result,we get mutant can bind BV,but the physical and chemical properties of Apc E mutant remain to be further optimized.Our work about ApcE can provide an advice for near-infrared fluorescent probe of phycobiliprotein.