The Molecular Evolution Of Near-infrared Fluorescence Proteins Based On The Study Of Phycobiliproteins And Lyases

Pigmentation of the light-harvesting phycobiliproteins of cyanobacteria requires covalent attachment of open-chain tetrapyrroles,bilins,to the apoproteins.With exception of ApcE,thioether formation via addition of a cysteine residue to the 3-ethylidene substituent of bilins is mediated by lyases.The mechanism research of lyase and property of biliproteins lay foundation for evolution of fluorescence protein based on biliproteins.T-type lyases are responsible for attaching bilins to Cys155 of phycobiliprotein b-subunits.We present the crystal structures of Cpc T?All5339?from Nostoc sp.PCC7120 and its complex with phycocyanobilin,at 1.95 and 2.50 ??? resolution,respectively.Cpc T adopts a calyx-shaped b-barrel fold,forming a dimer in the crystal lattice.Based on the structure and mutational analysis,a reaction mechanism is proposed that accounts for stabilizing the chromophore,and for regio-and stereo-specificity of the addition reaction.The chromophore is largely buried in the dimer but in the monomer the 3-ethylidene group is accessible for the apo-phycobiliprotein.At the dimer interface,a loop extending from one subunit partially shields the opening of the phycocyanobilin binding pocket in the other subunit.Deletion of the loop or disruptions of the dimer interface significantly reduce Cpc T lyase activity,suggesting functional relevance of the dimer.Dimerization is further enhanced by chromophore binding.The Cpc T lyase reaction is inhibited not only by thiols but also GSSG,which demonstrate the dynamic change of disulfide bond is essential for the catalytic activity.Asp163 and Tyr65 at the b-and a-face near the E-configured ethylidene group,respectively,support the acid-catalyzed nucleophilic Michael addition of cysteine-155 of the apoprotein to an N-acylimmonium intermediate.Such specificity may be guided by electrostatic interactions between charged surface of CpcB and CpcT.Phycobilisomes,the light-harvesting antennas in cyanobacteria and red algae,consist of an allophycocyanin core that is attached to the membrane via a core-membrane linker,and rods comprised of phycocyanin and often also phycoerythrin or phycoerythrocyanin.We here report the construction of a unique water-soluble loop-modified variant of core-membrane linker,Apc E??.The variant could still be covalently chromophorylated with both phycocyanobilin and phycoerythrobilin as wild type.The chromophorylated variant was trimer in the normal buffer,while it was dimer under the buffer containing 4 mol/L urea as wild type.While the trimerized variant chromophorylated with phycocyanobilin had a blue shifted absorption(?_max = 625 nm)and fluorescence(?_max = 650 nm),the dimerized variant chromophorylated with phycocyanobilin had the similar absorption(?_max = 659 nm)and fluorescence(?_max = 666 nm)as wild type.The change of spectra is also sensitive with temperature.The unusual spectral variations could be related to the structural changes accompanied with the oligomerization changes,which is investigated with stopped-flow absorption,fluorescence and circular dichroism.Recently,some cyanobacteria capable of far-red light photoacclimation?Fa RLi P?can use the far-red light??= 700-750 nm?long believed not used by cyanobateria to perform oxygenic photosynthesis.A gene cluster of photosystem core subunits controlled by a red/far-red phytochrome Rfp A was found in these cyanobacteria.ApcE2 and ApcF2 without the conserved cysteine were found when the sequence of phycobiliproteins in Fa RLi P gene cluster was analyzed by phylogenetic tree and alignmemnt.When expressed in E.coli with enzymes generating PCB,the chromoprotein PCB-Apc E2 and PCB-ApcF2 absorb at 700 nm and 673 nm,fluoresce at 714 nm and 700 nm,which were ⁴0nm and 56 nm red-shifted compared with canonical ApcE1 and ApcF1.The denatured spectra with 8 mol/L acid urea demonstrated the red-shifted spectra were induced by non-covalent binding of chromophore.This study provided the material for the evolution of far-red and near infrared fluorescence protein.Biliproteins have extended the spectral range of fluorescent proteins into the region of maximal transmission of most tissues and are favorable for multiplexing,but their application presents considerable challenges.Their fluorescence derives from open-chain tetrapyrrole chromophores which often require the introduction of dedicated reductases and lyases.In addition,their fluorescence yield generally decreases with increasing wavelengths,and depends strongly on the state of the binding protein.The reported biliproteins,termed BDFPs,are derived from the phycobilisome core subunit,ApcF2.This subunit is induced in the thermophilic cyanobacterium,Chroococcidiopsis thermalis,by far-red light and binds phycocyanobilin non-covalently.The BDFPs obtained by molecular evolution of ApcF2 bind the more readily accessible biliverdin covalently while retaining the red-shifted fluorescence in the near-infrared spectral region?⁷10 nm?.They are small monomers?¹5 k Da?and show not only excellent photostability,but are also thermostable up to 80 ?,tolerate acid down to pH 2,and high denaturant concentrations.The result indicates far-red adapting cyanobacteria as a useful source for designing extremely red-shifted fluorescent markers.In vivo performance of BDFPs as biomarkers in conventional and super-resolution microscopy,alone or fused to target proteins,is exemplified in several mammalian including human cell lines,in the nematode,Caenorhabditis elegans and,at low pH,in Lactobacillus lactis.