Study On Assemblies Of Pbss,Structure Of L_CM PB-Domain,And Function In State Transition Of Synechocystis Sp.PCC6803

The phycobilisomes(PBSs)of cyanobacteria and red-algae are unique megadaltons light-harvesting protein-pigment complexs that utilize bilin derivatives for light absorption and energy transfer.Recently,the high-resolution molecular structures of red-algae PBSs revealed how the core-membrane linker(L_CM)specifically organizes the allophycocyanin(APC)subunits in phycobilisome's core.However,the topology of L_CM in red-algae PBSs was different than the one suggested for cyanobacterial PBSs based on lower resolution structures.Particularly,the model for cyanobacteria assumed that the Arm2 domain of L_CMconnects the two basal APC cylinders in PBSs core,extending to the thylakoid membrane,and can interact with it.Whereas the red-algae PBSs structures revealed that Arm2 is partly buried in the core of one basal cylinder and connects it to the top cylinder.Here,we perform a biochemical analysis of mutations in the apc E gene that encodes L_CM from Synechocystis sp.PCC6803 and find that the L_CM topologies are actually the same in cyanobacteria and red-algae.We found that removing the top cylinder linker domain in L_CM splits the PBSs core longitudinally into two separate basal cylinders.Deleting either all or part of the helix-loop-helix domain at the N-terminal end of Arm2,disassembled the basal cylinders and resulted in degradation of the part containing the terminal emitter,Apc D.Similarly,deleting the following 30 amino-acid loop seriously affected the assembly of the basal cylinders and leaded to a large number of phycocyanin(PC)degradation.However,further deletion of the amino-acids at the C-terminal half of Arm2 had only minor effects on the assembly of PBSs.Altogether,the biochemical data is consistent with the red-algae L_CMtopology suggesting that the PBS cores in cyanobacteria and red-algae assemble in the same way.It provides new directions and ideas for the study and enables us to know more about Synechocystis sp.PCC6803 PBSs.In order to further monitor the state transition in these mutants above,we perform a pulse amplitude modulated fluorometer(PAM-2500)and 77 K fluorescence technique on wild-type and mutants.The results showed that the mutants removing Rep3 domain could perform state transition while the mutants having the damaged helix-turn-helix element were incapable of state transitions.Damaging the other Arm2 domain only disturbed the state II transition but not interfered with the state I transition.The state transition had no connection with PBSs rods.It is indicated here that state transitions result from the longitudinal movements between the two halves of every cylinder in the PBSs core,which are connected by Arm2,or are related to the integrity of basal cylinder.Only such state transition model can explain the results reported here.This study breaks our tether with?PBSs movement?and?spillover?models,enriches the state transition models and offers a better guide to the future research.The PBSs are assembled and perform state transition via L_CM,while the PB-domain of L_CM is one of the terminal emitters helping PBSs transfer energy to light reaction center.Many researchers focused on the PB-domain of L_CM to explore the structure.Here we focus on a variant of PB-domain from Synechococcus sp.PCC7335 L_CM(Apc E2)that has the best solubility and retains spectral red-shift.The crystal structure of the protein(named BDFP3.2)was resolved at 2.8(?),but the loop domain and PCB chromophore were invisible.In the future,we need to improve crystal resolution:changing crystallization conditions,reusing random mutagenesis or site-directed mutagenesis to screen out gene fragments.If the resolution is enough,we will resolve the first structure of PB-domain containing Loop domain and non-covalently binding PCB.