| Phycobilisome（PBS）is a large light-harvesting complex in cyanobacteria,which can absorb light energy and transmit it to the photosystems.The core and rod of PBS are composed of linker proteins and phycobiliproteins（PBPs）bound with phycobilin,which then spontaneously assembles into a highly ordered complete PBS complex.PBS complex will degrade dynamically during the period of light and nutrition deficiency,along with serious"bleaching"phenomenon,and will be remolded after the nutrition condition is supplemented.In previous studies,non-bleaching（Nb）proteins were associated with the degradation of phycobilisomes,including NblA and NblB.In this study,phycobiliproteins and tool enzymes of phycobiliprotein from Nostoc sp.PCC7120 were tested in vitro.The tool enzymes included three three kinds of lyases,NblA and NblB.The experimental results are as follows:1)NblA and NblB promote the degradation of the whole PBS and PBS core,especially allophycocyanin（APC）.2)Cpc E/F,Cpc S and Cpc T as biosynthetic enzymes,have more wider functions than the canonical lyases.The catalytic action of lyases is reversible:they not only bind PCB to PBPs,but also remove and transfer PCB to other apo-biliproteins without PCB.3)NblA and NblB can modulate lyase-catalyzed binding and detachment of chromophores in a complex fashion.In this process,NblA will inhibit the chromophorylation and chromophore transfer reactions of Cpc E/F,while NblA and NblB will enhance the catalytic effect of Cpc T.4)PCB will be transferred between PBPs with lyase-catalyzed and lyase-independent,and NblA/B will regulate this process.5)PBPs and lyases interact with NblA and NblB.Although there is no interaction between NblA and NblB,both NblA and NblB interact with lyase and PBPs（including apo-biliproteins）,the only phycobiliprotein that has no interaction with NblA or NblB is Apc EΔ(Soluble domain of core-membrane linker protein-Apc E,LCM).In conclusion,this study confirms that the degradation and remodeling of PBS are induced by NblA and NblB after changes in the surrounding environment,and the specific action site of NblA and NblB in the degradation of phycobilisome is determined as APC.Meanwhile,for the first time in this study,it was found that all three lyases can catalyze the separation of phycobiliproteins and even phycobiliosome chromophores（PCB）,and PCB undergo dynamic transfer between PBS subunits.Furthermore,the study found that the pigment transfer process is regulated by NblA and NblB.In addition,The study first found that phycobiliproteins（excluding Apc EΔ）and lyases all interact with NblA/B.In order to construct an artificial photosynthetic element with the function of capturing light and transmitting energy,holo-phycobiliproteins have been successfully synthesized,and the techniques and methods of molecular design and artificial assembly of phycobiliproteins have been established.On this basis,this study will use the phycobiliprotein Apc F2 as a template for molecular evolution to obtain the Beidou fluorescent protein for molecular design and optimization in order to build a light-harvesting and energy-transfer element that can transfer energy to the photosystem.First,we combine BDFPs with PEB for chromophorylation then calculate the chromophorylation rate,molar extinction coefficient and fluorescence quantum yield through the analysis of the spectra,and BDFP2.2 and BDFP2.3 were selected for a series of improvements,including adding SA and GCN4 domain connector to BDFP2.2/2.3,improving its spectral properties.A system of co-expression of BDFP2.2/2.3 and Encap was constructed,and a fusion protein of about 1MD was formed,then FRET phenomenon occurs when additional PCB is added. |
