Research On A Kind Of Intinsically Disordered Protein-Soybean PM1 Protein And Its N/C Peptides Which Could Interact With DNA

Late embryogenesis abundant proteins?LEA protein?are Intrinsically disordered proteins?IDPs?.They play an important protective role in improvingstress tolerance of plant.Many evidences demonstrate that LEA proteins can protect proteins?enzyme activity?,lipids and cell membrane structure under stress and also have the ability to bind metal ions.However,the evidences about interaction of LEA proteins with DNA molecules are still rare.The PM1 protein of soybean belongs to the fourth group of LEA protein and they can be located in both the nucleus and cytoplasm.In the present paper,we are focusing to learn the interactions between PM1?PM1-N/PM1-C?proteins and five different DNA molecules?PM1,EGFP,HSP70,pGL3 andpYES2?and also the influence of several metal ions(Zn²⁺,Mg²⁺,Ca²⁺,Ni²⁺,Mn²⁺and Cu²⁺)and pH values?6.3,8.0,and 10.2?on PM1?PM1-N/PM1-C?-DNA interactions.Electrophoretic mobility shift assay?EMSA?was used firstly.The results showed that PM1 protein can interact with five DNA molecules after incubatingthe PM1 proteinwith five kinds of DNAand then subjecting to agarose gel electrophoresis.Then the results showed that six metal ions had not apparent influence on the interactions between PM1?PM1-N/PM1-C?proteins and five DNA molecules when mixed with PM1 protein and the five kinds of DNA.Furthermore,Filter binding assay?FBA?was used to investigate the interactions of PM1?PM1-N/PM1-C?and DNA molecules.Two kinds of DNA fragments?PM1 and pGL3?were selected to labele with fluorescein isothiocyanate?FITC?.Thenthe following researches were carried out:1)The PM1?PM1-N/PM1-C?protein were mixed with two FITC-labelled DNA fragments,respectively.The FBA results showed that the PM1?PM1-N/PM1-C?protein could bind withtwo kinds of DNA fragments.2)The PM1?PM1-N/PM1-C?protein was mixed with six metal ions and then DNA.The results showed that fourkinds of metal ions(Zn²⁺,Mg²⁺,Ca²⁺and Mn²⁺)had no apperentinfluence on the interactions between PM1?PM1-N/PM1-C?proteins and five DNAmolecules.However,the two metal ions(Ni²⁺and Cu²⁺)couldobviously weak their interactions.3)On the other hand,when PM1?PM1-N/PM1-C?protein was mixed with DNA firstly and then six metal ions were added,the FBA results showed that the fourmetal ions(Zn²⁺,Mg²⁺,Ca²⁺and Mn²⁺)had not apperant effect on the interaction of PM1?PM1-N/PM1-C?proteins with DNA,whereas Cu²⁺and Ni²⁺could attenuat their interactions,the weakening extent of Ni²⁺was not as good as Cu²⁺.4)The three p H values had no effect on their interactions.Circular dichroism analysis was used to study the effect of two DNA fragments?PM1andpGL3?on the secondary structure of PM1?PM1-N/PM1-C?proteins.The results showed that the secondary structure of PM1?PM1-N/PM1-C?proteins are mainly random coils;when PM1?PM1-N/PM1-C?proteins and DNA were incubated together,there was not significant change in protein secondary structureof the proteins.In summary,1)PM1?PM1-N/PM1-C?protein can interact with differentDNA,which belongs tothe non-specific binding.2)Both Ni²⁺and Cu²⁺couldweakthe interactions betweenPM1?PM1-N/PM1-C?proteins with DNA to some degree;Ni²⁺,Cu²⁺and PM1 can bind competitively with the DNAmolecules.3)pH values have noinfluence on the interactions between PM1?PM1-N/PM1-C?protein and DNA molecules.4)DNA couldn't affect the secondary structure of PM1?PM1-N/PM1-C?proteins.