The Study Of Bright Monomer Far-red And Near-infrared Phycobiliprotein Fluorescent Probes

Biological tissue has an optical "transparent window" with a spectral range of650-900 nm,because cellular components such as the hemoglobin,water,lipids and melanin in this region is to minimize the absorbance and allow light to penetrate tissues more efficiently.In addition,lower autofluorescence and light scattering make fluorescent protein in this spectral region suitable for deep-tissue imaging.At present,the spectral range of biliproteins have been extended to the far-red regions(FR,650-700 nm)and near-infrared regions(NIR,700-770 nm).FR and NIR FPs are important for relevant bioimaging and multiplex labeling.However,with the increase of emission wavelength of FPs,fluorescence quantum yield and fluorescence intensity generally decrease correspondingly.Therefore,it is still a considerable challenge to maintain the emission in near-infrared regions while ensuring the high brightness and monomer state of FPs.Two FR-induced phycobilisome core subunits,ApcE2 and ApcF2,are found in FR-adapting cyanobacteria.ApcE2 is from Synechococcus sp.PCC7335 and ApcF2 is from Chroococcidiopsis thermalis sp.PCC7203.The soluble BDFP3(Bei Dou Fluorescent Protein)domain,engineered from ApcE2,binds phytochromobilin non-covalently,termed BDFP3.1.It absorbs at 708 nm and fluoresces at 720 nm.P?B was added externally;it is obtained by the purified BDFP3.1 under denaturing conditions and subsequent extraction.Then We fuse two BDFP1.1 with one BDFP3 to design a fluorescent biliprotein triad,termed BDFP1.1:3.1:1.1,which has an emission wavelength of 722 nm and exists as a monomer.It fluoresces 2.7-fold brighter than that of the most red-shifted phytochrome,IRFP720.BDFP1.1 domain,engineered from ApcF2,covalently binds biliverdin and fluoresces at 707 nm.In this study,the fluorescence intensity of covalently bound BV of BDFP1.1 is effectively combined with the red-shift spectrum of non-covalently bound P?B of BDFP3,and the energy of BDFP1.1-BV(as donor)is transferred to BDFP3.1(as receptor)by fluorescence resonance energy transfer(FRET),thereby increasing the fluorescence intensity at 720 nm.Labeling of different target proteins by fusion to the biliprotein triad is well demonstrated in prokaryotic and eukaryotic mammalian cells.Inspired by BDFP1.1:3.1:1.1 triads,we then design two new fluorescent biliprotein triads,respectively BDFP1.2:3.3:1.2 and BDFP1.6:3.3:1.6.In mammalian cells,these triads have maximal emission wavelengths of 670 nm and not only have an unprecedentedly brightness in the FR region,but also exist as monomers.The soluble BDFP3 domain binds phycoerythrobilin non-covalently,termed BDFP3.3.It is a bright red fluorescent protein with maximal absorbance at 608 nm and fluorescence at 619 nm,and the fluorescence quantum yield can reach 66%.Because only biliverdin exists in mammalian cells,the chromophore PEB needs to be added externally to bind to BDFP3 when expressed in mammalian cells.The extraction of PEB can be replaced by proteolytically digested BDFP3.3.BDFP1.2 and BDFP1.6 domains are derived from ApcF2 and covalently bind to BV.For BDFP1.2:3.3:1.2,the red-shift spectrum of covalently bound BV of BDFP1.2 is effectively combined with the fluorescence brightness of non-covalently bound PEB of BDFP3,and the energy of BDFP3.3(as donor)is transferred to BDFP1.2-BV(as receptor)by fluorescence resonance energy transfer(FRET),thus improving the fluorescence brightness at 670 nm.The design concept of BDFP1.6:3.3:1.6 is similar.In vivo performance of these new biliproteins as biomarkers in super-resolution microscopy,fused to target proteins,is well demonstrated.The phycobiliprotein-derived BDFP3.3 and BDFP1.2/1.6:3.3:1.2/1.6 are the first to realize dual-color labeling in red and far-red regions,and the method to increase the effective brightness by triads also provides a new idea for the development of more excellent fluorescent proteins.BDFPs are derived from FR light-induced allophycocyanin ?-subunit(ApcF2)from Chroococcidiopsis thermalis sp.PCC7203 and can covalently bind to BV.Far-red BDFPs and near-infrared BDFPs maximally fluoresce at ?670 nm and ?710 nm,respectively.With low molecular weight,high solubility and red-shift spectrum,BDFPs are ideal templates for the evolution of excellent near-infrared fluorescent probes.Using BDFP1.2(32-169)as the molecular evolution template,we obtained a variant of far-red BDFP and resolved the crystal structure.The structure shows that one BV chromophore is doubly covalently bound with both Cys72 and Cys82.Based on the structure,we further optimized a series of monomeric far-red and near-infrared fluorescent proteins BDFPs.These newly-developed BDFPs are remarkably brighter than the formerly reported far-red and near-infrared monomeric fluorescent proteins and are more suitable as biomarkers for bioimaging.