| Light,as a near-ubiquitous source of energy and information,is used in two major ways: being converted into chemical energy for organisms via photosynthesis and adjusting organism physiological process with photoreceptors.Phycobiliproteins that bind bilins are organized as light-harvesting complexes,phycobilisomes,in cyanobacteria,the model organism of photosynthesis.Cyanobacteria have evolved many mechanisms to maximize their photosynthetic efficiency,including remodel the phycobilisomes in response to the incident irradiation with cyanobacteriochrome.In the phycobilisomes,core-membrane linker(ApcE),the terminal of energy transfer with more red-shift spectrum than the other phycobiliproteins is advantageous to evolve into the near-infrared fluorescent probe.While cyanobacteriochrome(CBCR)respond to changes of light is suitable for development of light-gated protein switches.In the normal cyanobacteria,covalently bound phycocyanobilin of ApcE1 absorbs near 660 nm and fluoresces near 675 nm.In cyanobacteria capable of near infrared photoacclimation,such as Synechococcus sp.PCC7335,there exist even further spectrally red shifted components absorbing >700 nm and fluorescing >710 nm.We found an extra core-membrane linker(ApcE2)without the canonical cysteine and cloned the chromophore domain(1-273)to the express vector,and expressed in E.coli together with enzymes generating the chromophore,PCB,PEB,PФB.The resulting chromoproteins,PCB,PEB,PФB-ApcE2(1-273),absorb at 700 nm,615 nm,711 nm and fluoresce at 714 nm,628 nm 726 nm,respectively,and red shift of ~40 nm compared with canonical ApcE1.The extreme spectral red-shift could not be ascribed to exciton coupling: dimeric PCB-ApcE2(1-273)and monomeric PCB-ApcE2(24-245)absorbed and fluoresced similarly.After a series of experiments,we proved that the red shift of the spectrum mainly resulted from non-covalent binding of the chromophore by which its full conjugation length including the Δ3,31 double bond is preserved.The self-assembled non-covalent chromophorylation demonstrates a novel access to red and near-infrared emitting fluorophores.Brightly fluorescent biomarking was exemplified in E.coli by single-plasmid transformation.No fluorescence was detected when ApcE2 expresssed and chromophored by add pigment in vitro or synthesis of pigment in vivo in animal cells.To be further optimized for a near-infrared fluorescent probe,ApcE2(24-245)was subjected to molecular evolution by site-directed mutagenesis,a mutant that could self-assemble with biliverdin non-covalently and led to a further red shift spectrum of absorption at 730 nm was identified.This work is a foundation for further optimization research.ApcF2 without the canonical cysteine is found in Chr.thermalis PCC 7203,capable of near infrared photoacclimation.We expressed ApcF2 in E.coli together with enzymes generating the chromophore,PCB.The resulting chromoprotein,PCB-ApcF2,absorbs at 674 nm and fluoresces at 700 nm,and also binds the chromophore non-covalently.Dimeric PCB-ApcF2 can be engineered into monomer after truncation of the N-terminal,which is also essential for maintaining activity of protein chromophorylation.Compared with ApcE2,ApcF2 has certain advantages for engineering near-infrared fluorescent probes.First,ApcF2 has smaller molecular weight so as not to perturb the stoichiometry of the protein of interest.Second,ApcF2 is soluble.Third,ApcF2 remains stable over a wider range of pH,which makes it potentially applicable under harsh conditions.In our study,ApcF2 was localized to the membrane in E.coli and showed obvious fluorescence,this work laid the foundation for the next step to mark in animal cells.Random mutagenesis was performed to ApcF2(20-169),we obtained a mutant with the blue-shift spectrum and a higher fluorescence quantum yield,which offers the raw materials for double color biomarking.In recent years,more and more researchers have focused on the design of optogenetic tools by phytochromes.For the opacity of mammalian tissue to visible light and the strong attenuation of infrared light by water at >900 nm have contributed to growing interest in the development of photoreceptors absorbing far-red and near-infrared.In this study,we applied molecular evolution to 1393GAF3 from Synechocystis sp.PCC6803,and tried to engineer a mutant,that could response to the far-red and near-infrared.Althought we didn’t get the ideal mutant,some key residues were found related to spectral tuning and remain the protein activity by analyzing the spectrum and mutation sites,which laid a foundation for further researches. |
