Optogenetics Study And Molecular Evolution Of Cyanobacteriochromes Slr1393g3

Photoreceptors such as cyanobacteriochromes could absorb a very wide range of light wavelengths which from near ultraviolet(~350 nm)to far-red(~750 nm),and convert to biological signals to regulate various signal pathways in living organisms.Photoreceptors are capable of controlling biological pathways in spatiotemporal precisely and sensitively which can act as a broad resource for the development of fluorescent proteins,biosensors,and optogenetic tools.Optogenetics has attracted tremendous attention due to its capability to control the "on-off" switching mode of biological pathways.In this study,we used a typical red/green light switch,Slr1393g3 which also named RGS(Red-Green switchable protein),from the cyanobacteria Synechocystis sp.PCC6803.RGS can covalently bind to phycocyanin PCB,which showed a red light-absorbing thermostable state absorption peak at 649 nm and a green light-absorbing metastable state at 535 nm.RGS was fused with a blue light receptor(LOV)-regulated Adenylyl Cyclase(AC)through the C-terminal ? helix of RGS to obtain a red/green-regulated adenylate cyclase.To make RGS acquire a regulatory capacity for AC,we conducted a series of truncation mutations in the linker region of RGS and AC.The catalytic activity of the obtained fusion protein RGS?14-?4AC was 2.2 times higher in green state than in red light state under in vitro conditions and it could up to 3 times when the reaction temperature raise to 35 °C.In E.coli ?cya(the gene encodes adenylate cyclase),the activity could show 10 times higher in green light than in red light when the fusion protein was expressed for 14 h in vivo.The fusion protein RGS?7-?4AC had 7 amino acids more than the RGS?14-?4AC at the C-terminus of RGS,which was exactly the length of an ?-helix,and its catalytic activity was 1.6 times higher in green state than in red state,which demonstrates that the ability of light to produce a conformational change in RGS was transmitted to the AC via the ?-helix.Although RGS without a strong capacity to regulate AC,it proves that CBCRs are feasible as optogenetic tools,laying the foundation for subsequent research on CBCRs.In recent years,the biliverdin BV linked CBCRs have been found in Acaryochloris marina.In this study,we conducted random mutagenesis combined with site-directed mutagenesis using Slr1393g3 as a template and acquired a series of mutated proteins that could bind to BV.The spectra of these mutants were converted from original red and green light responses to the far-red and red light responses.Among them,the Variant 4-1 was the most significant,whose absorption peak in far-red Pfr ground state was at 695 nm,and the absorption peak in red Pr excited state was at 635 nm.Based on the structure simulation,the mutation of F474 I was especially important for the BV binding.Phe was originally located near the D ring of PCB,and the D ring of BV extended more outward than PCB,thus the side chain of Phe hindered the BV binding.Asp532 also had a significant effect on the BV binding,and this site mutation caused a special change in the spectrum.Although our study did not obtain a perfect mutant to bind BV,the results obtained from the PCB to BV binding of CBCRs can guide other mutagenesis transformation of CBCRs.phycobilisome(PBS)in cyanobacteria is a large complex composed of multiple subunits located at the membrane matrix of thylakoid.On the condition of nitrogen or sulfur deficiency,PBS showed bleaching effect due to the synthesis inhibition and degradation of phycocyanin and allophycocyanin,and caused cyanobacteria cells to turn from blue-green to yellow-green.NblA and NblB were thought to mediate the degradation of PBS.NblB has a high sequence homology with CpcE,a component of lyase CpcE/F,and NblB combined with CpcF can play the functions similar to CpcE/F,although the effect is weak.By adding NblA and NblB to the reaction system of CpcE/F,it was found that NblA inhibited the catalytic function of CpcE/F,and the inhibitory capacity of NblA is positive correlation with its concentration,whereas the catalytic function of CpcE/F was slightly enhanced by NblB.The above results indicated that the functions of NblA and NblB may not be limited to the degradation of PBS,but also in other biological pathways in cyanobacteria.