Study On The Assembly And Assembly Stability Of Agrobacterium Tumefaciens Bacterioferritin In Vitro

As an essential element for organisms,iron is involved in a variety of important physiological and biochemical processes,acting as a cofactor for proteins.Iron deficiency affects growth,metabolism,and the virulence of pathogenic bacteria,while excess iron can trigger the Fenton reaction,causing oxidative damage to cells.Ferritin is able to use O2 or H2O2 to oxidize ferrous iron to ferric iron and store it in the internal cavity.Ferric iron can be reduced and released by ferritin when cells are iron deficient.Therefore,ferritin plays an important role in maintaining cellular iron homeostasis and relieving iron toxicity.Apart from that,ferritin is also considered a good nanomaterial.Ferritin is a 24 or 12 polymer with the shape of a hollow sphere that is chemically and thermally stable in vitro and can be controlled in its assembly and de-assembly by changing environmental conditions.Ferritin is also resistant to modification of its outer surface,inner cavity,subunit-interacting interfaces,etc.It has great potential and value for biomedical,chemical,and biological nanotechnology applications.Bacteria generally contain three ferritins:classical ferritin(Ftn),bacterioferritin(Bfr),and DNA-binding protein from starved cells(Dps).Ftn and Bfr are 24-mer proteins and Dps is a 12-mer protein.Most bacteria have both Ftn and Bfr or two kinds of Bfr,whereas Agrobacterium tumefaciens has only one Bfr(AtBfr)and one Dps(AtDps).Unlike Bfr in other bacteria,AtBfr has eight extra amino acids at its Nterminal end,the possible effect or role of which is unclear.In Agrobacterium tumefaciens,Bfr is the main functional ferritin storing iron.The function of ferritin is closely related to its structure,and studying the self-assembly of ferritin in Agrobacterium tumefaciens provides a theoretical basis for the application of ferritin and is also important for the study of the function and mechanism of ferritin.In this study,we constructed deletion and complementation strains of ferritin genes and identified the correct reading frame of the bfr gene in Agrobacterium tumefaciens.A variety of recombinant ferritins from Agrobacterium tumefaciens were purified;the buffering system of ferritin in vitro was optimized;the effect of key amino acids on the assembly and function of bacterioferritin from Agrobacterium tumefaciens was investigated,and the effect of divalent metal ions and heme on its assembly was explored.The main results are as follows:(1)Gene deletion and complementation strains of Agrobacterium tumefaciens ferritin were constructed.Trivalent iron-specific staining experiments found that the bfr gene of Agrobacterium tumefaciens open reading frame starts at AUG,encoding 169 amino acids instead of 161 amino acids.(2)The self-assembly of ferritin in different buffers,pH,and salt concentrations was observed by Native PAGE to optimise the buffering system of Agrobacterium tumefaciens ferritin in vitro.It was found that AtDps could form a stable 12-mer in vitro,while AtBfr was a mixture of 24-mers and oligomers.AtBfr had the optimal polymerization state in 20 mM HEPES(pH 8.2)buffer containing 50 mM NaCl.(3)The polymerization state and structural stability of AtBfr recombinant proteins with N-terminal and C-terminal His tags were observed in vitro using differential fluorescence scanning,and Native PAGE.The results showed that AtBfr with Nterminal 6×His tag could only form oligomers,while AtBfr with N-terminal long His tag and C-terminal 6× His tag could form 24-mers with similar thermal stability,but the proportion of 24-mers in AtBfr protein with C-terminal 6× His tag was higher.(4)Two point mutant proteins,AtBfrM60L and AtBfrE74R were constructed.The structure and assembly of the mutant proteins were investigated using circular dichroism,transmission electron microscopy,differential fluorescence scanning and Native PAGE.The results showed that,though the amino acid mutations at these two sites did not affect the secondary structure of Bfr,they had a significant effect on its thermal stability and the assembly of nanocages.The thermal stability and the proportion of 24 polymers of AtBfrM60L decreased,and the size of the protein cage formed was smaller,while the thermal stability and the proportion of 24 polymers of AtBfrE74R increased.The protein cage formed by AtBfrE74R was similar to that of WTAtBfr,but the overall particle size distribution was more concentrated.(5)The effects of different divalent metal ions and heme on AtBfr were examined.The effects of different divalent metal ions on the thermal stability of AtBfr were observed by differential scanning,and the results showed that Fe2+,Mg2+,and Mn2+had no significant effects on WT-Bfr,but improved the thermal stability of AtBfrM60L and AtBfrE74R,while Zn2+treatment resulted in significant changes in the melt temperature of all recombinant AtBfr proteins,which showed a decrease in the Bfr protein monomeric structure stability.(6)The heme content of WT-AtBfr and AtBfrM60L was determined,and the effect of heme binding on the aggregation of AtBfr proteins was investigated by means of heme reconstitution.It was found that the mutation of amino acid 60 resulted in the inability of AtBfr to bind heme,whereas the increased heme binding in WT-AtBfr facilitated the self-assembly of the protein in vitro.