Study On Enzyme Activity Protection And Interaction Proteins Of Soybean PM18?/? Protein

Intrinsically disordered protein(IDP)fails to form stable secondary or tertiary structure,yet they exhibit biological activities.IDP plays important roles in molecular regulatory network,many IDPs undergo disorder-to-order transitions to performs various functions such as molecular chaperone,site modification,molecular effector,molecular assembly and detoxification.Alternative splicing(AS),which usually occurs in disordered region of proteins under various stresses,is an important way for regulation of gene expression,as well as a basis for the production of diverse functionally proteins in eukaryotes.AS plays an important role in regulation of enzyme activity,subcellular localization,and post-translational modification of proteins.Therefore,the mechanism of how IDPs participate in stress response through alternative splicing in plants is worth studying.Late embryogenesis abundant(LEA)is a type of IDP which are closely related with plant stress resistance.The LEA proteins can be induced to express by abiotic stresses such as drought,high temperature,and high salt stress.LEA proteins could maintain protein activity,inhibit protein aggregation,stabilize protein conformation and protect membrane structure under stress.Soybean PM18 belongs to LEA3 protein and there are two transcripts-PM18? and PM18?.In our previous study,PM18? and PM18? proteins were identified in the heat-stable proteins of soybean radicle.However,the mechanism of PM18 gene and its alternative splicing in stress resistance has not been reported.In this study,bioinformatical analysis of two transcripts of soybean PM18 was analyzed.Both PM18? and PM18? were predicted to be intrinsically disordered proteins by the ANCHOR and PONDR.Both two proteins shows high degree of disorder at the N-terminus and C-terminus.PM18? was longer than PM18? at the domain in the different amino acid positions,but shorter at the C-terminus.PM18? was predicted to have more helical and functional binding sites than PM18? at the C-terminus by the PREDICT PROTEIN,SMART.Both PM18? and PM18? were predicted to locate in the nucleus or cytoplasm by BaCeILo and SignalP4.1.By modeling the protein structure,PM18? is consisted of three helical structures,while PM18? shows two helical structure by SWISS-MODEL.To compare the structural and functional differences between PM18? and PM18?,the prokaryotic expression vectors of PM18? and PM18? were constructed,and recombinant proteins were purified by affinity chromatography.The results of circular dichroism spectra showed that they were all disordered proteins.In the presence of 25% TFE and 1 mM SDS,both of PM18? and PM18? could form more than 80% alpha helix structure.The conformation of PM18? could formed stronger ?-helix structure while PM18? would like to form ?-sheet structure in the presence of PEG.PM18?(1.8 ?M)could keep the LDH residual enzyme activity above 70% under freezing and thawing stress,while the the residue enzyme activity reached to about 90% under the protection of PM18? at the same concentration.PM18 protein protected LDH enzyme under 55°C stress at high concentration(1.8 ?M).At the same concentration,the PM18 protein could not protect LDH enzyme in the presence of Dexteran(80g/L),but both of them showed a decrease in protective effect with PEG.However,in the presence of Ficoll with 55°C stress,LDH could be protected by the PM18? protein,while the PM18? protein had no protective effect.It showed that different macromolecular crowding agents had different effects on the protection of LDH enzymatic activity by PM18.Taken together,there was little difference in the structure and function of PM18? and PM18? in vitro.IDP could bind different proteins to paricipate in different physiological process.The mixed cDNA library of soybean roots and leaves was screened for the interacting proteins of PM18? protein by yeast two-hybrid,and 30 positive clones were found to be positive.Moreover 6 proteins were found to be interact with PM18? before.After the rotary validation and sequencing,11 candidate proteins were obtained.Compared with the interacting proteins of PM18?,4 proteins were the same or belonged to the same family.PM18?-1 and PM18?-8 belong to the voltage-dependent anion channel protein family(VDAC),PM18?-17 belongs to the plastocyanin family protein,PM18?-27 and PM18?-9 belong to the MYB transcription factor family.PM18?might interact with methionine aminopeptidase family,histone H2 A,HSP40 family and AP1 GI protein family.In a word,PM18? and PM18? may interact with different proteins.In summary,the two transcriptional isomers of PM18 gene show a few differences in structure and function in vitro.Moreover,they may interact with different proteins to participate in various regulating pathways.These results provided new evidence for the research of stress resistance mechanism of PM18 gene.