Crystal Structure Of PSB27 In Arabidopsis Photosynthesis System ?

The Arabidopsis thaliana PSB27(AtPsb27)protein is located in the thylakoid lumen,and the PSB27 protein is encoded by the nuclear gene At lg03600,The molecular weights of the mature protein is 11.7KD.The N-terminal leader peptide sequence consists of 67 amino acid residues that help the PSB27 protein enter the thylakoid lumen.PSB27 is essential for plants to adapt to variable light intensity.The mutant in constant light or low light,and compared with the wild type Arabidopsis phenotype have no significant difference;but in the treatment of abnormal growth under variable light,the mutant is very small,the mutant was due to the deletion of psb27 gene.In view of the fact that psb27 mutants are specifically sensitive to altered light,it is speculated that PSB27 protein is one of the key factors for plant to respond to changing light.We suspect that the pH of the thylakoid lumen may change with the changing light intensity,and the acidic or basic amino acids in the protein may induce different pH.In contrast,the different pH of the thylakoid lumen environment will also affect the structure of PSB27.The structure of PSB27 was analyzed by X-ray diffraction.Based on the crystal structure,the Arabidopsis PSB27 isotope 15N labeled protein was further applied to heteronuclear single quantum coherence(Heteronuclear single quantum coherence,HSQC)spectroscopy.And the main and side chains of the 15N and 13C-labeled PSB27 proteins in the solution state were identified by nuclear magnetic resonance(NMR)techniques.In addition,protein and sequence analysis revealed that the acidic and basic amino acid residues between the alpha helices were very special in the plant,and these amino acids could be very sensitive to the external environment and alter the protein structure.So we will turn them into neutral amino acids and analysis of the structure through circular dichroism.The structural analysis of the PSB27 protein will allow us to understand the response mechanism of plants to changes in light intensity and is of great significance on the study of photosynthesis.This study mainly carried out three aspects of research,to obtain the following main results:Firstly,the recombinant plasmid pGEX.4T-2-PSB27 was constructed and sequenced,and then used for the expression and purification of PSB27 protein.The recombinant protein was mainly expressed in soluble form,and the GST tag of PSB27 was cleaved with TEV The protein was separated and purified by molecular sieve to obtain high purity protein,concentrated to the appropriate concentration for protein crystallization test,and then sent to Shanghai Synchrotron Radiation Source(SSRF)BL17U line station to do x-ray diffraction and data collection.We determined the crystal structure of Arabidopsis Psb27 with a resolution of 1.85A.X-ray diffraction results showed that PSB27 had four a-helices:H1(residues 3-22),H2(residues 30-49),H3(residues 58-77)and H4(residues 85-104),H2 And H3 have a small alpha helix(residue 52Arg-56Gly,designated as H*),similar to the structure of cyanobacteria Psb27.However,there are several structural differences between AtPsb27 and its blue bacterial homologues,mainly at the N and C-terminal surface charges.These differences indicate that PSB27 in higher plants and cyanobacteria can play a role in different working mechanisms.Thylakoid is an acidic environment with a pH range of 4.5-7.8,in order to test whether the pH changes in the thylakoid lumen affect the conformation of AtPsb27,the1H-15Nradio-labeled AtPsb27 samples were analyzed by the heteronuclear single quantum coherence(HSQC)spectrum over a range of pH values from4.5,5.5,6.7,to 7.5.W e found that the AtPsb27 protein aggregated under an environment at pH below 5.5 in a buffer containing 20mM PBS,250mM NaCl,2.5mM DTT,and 2.5mMNa2EDTA,suggesting AtPsb27 is not stable and confirmation is affected by a lower pH environment.The AtPsb27 protein sample is well stable at pH 6.7,and applied to HSQC spectrum analysis.The HSQC spectrum of 0.4 mM AtPsb27 recorded at pH5.5,6.7 and 7.5 are almost identical,The resonance signals are well-dispersed,indicating the protein was folded very well.AtPsb27 protein samples were particularly stable at pH 6.7 and could be used to identify the main and side chains of the 15N and 13C-labeled Psb27 proteins in solution.Some studies have shown that Psb27 in T.elongatus is located on the surface of PSII and the interaction of PSB27-K91 with CP43-K381 by cross-linking method.The residue 91K92L93K of PSB27 in T.elongatus corresponds to the 86K87R88K residue on H4 of AtPsb27.And 373LSRLKKDIQPWQERR391 of AtCP43 was similar to the peptide fragment of 381KNDIQPWQERR391 in T.elongatus CP43(TeCP43).We want to test the interaction between T.elongatus Psb27(TePsb27)and CP43 would applied to the AtPsb27 and CP43.We synthesized a stretch of amino acid sequence,377LSRLKKDIQPWQERR391 of AtCP43 similar to TeCP43,and use HSQC titration approach to test the interaction between AtPsb27 and the peptide fragment of CP43.However,the HSQC spectra of AtPsb27 were almost identical after addition of various concentration of the small peptide of 377LSRLKKDIQPWQERR391,means the small peptide does not affect the confiramtion of AtPsb27.It is not likely there is a direct interaction between AtPsb27 and this small peptide of AtCP43.Since only a small fraction of CP43 protein was applied to the test,we could not exclude the possible interaction between At Psb27 and AtCP43.Finally,a recombinant plasmid of wild type pGEX.4T-3-PSB27 was constructed.The plasmid was used as the template to construct the PSB27 point mutation vector,and then the protein expression and purification of the PSB27 mutant were performed,The secondary structure was analyzed by circular dichroism,Circular dichroism analysis experiment is still in progress.