Investigationg Of Chaperone-like Function Of The LEA3 Hydrophilic Domain Under Stress Conditions

Late embryogenesis abundant(LEA)proteins,found in many plant species and micro-organism,can protect cell from damage as a chaperone.LEA proteins are classified into seven groups.Among them,group 3 LEA proteins commonly contain an hydrophilic domain(HD)composed of conserved motif.Different HDs from various organisms contain different motifs,however,the function those hydrophilic domains are not fully understood.In this study,4 HDs of LEA3 were isolated from Brassica napus,Yarrowia lipolytica CLIB122,Caenorhabditis elegans and Deinococcus radiodurans,named Bnhd,Ylhd,Cehd and Drhd,respectively.This study mainly focused on Chaperone-like functional investigation of these HDs under several stress conditions,and major findings and progress are listed as below:1.Sequence analysis and structure prediction indicated that the grand average hydropathy(GRAVY)value of Bnhd was-1.487,the GRAVY value of Ylhd was-1.107,the GRAVY value of Cehd was-1.294,and the GRAVY value of Drhd was-1.029.In addition,protein disorder prediction suggested these 4 proteins are all disordered proteins.Therefore,the results demonstrated the four HD proteins have the similar characteristics of high hydrophilicity and complete disorder.Furthermore,Cehd contains 24 motifs,Ylhd contains 12 motifs,Drhd contains 8 motifs,and Bnhd contains 6 motifs,revealing that HDs contain different number of motifs in various organisms.2.In order to study the secondary structure of HD,CD spectra analysis was used to investigate the structure features of HD proteins.The result showed that all the HD proteins stayed at disordered state in normal solution.When added glycerol,which can be used to simulate the drought conditions,or 2,2,2-trifluoro ethanol(TFE),an ?-helix inducer,the secondary structure of hydrophilic domain changed to the structure that is rich in ?-helix.The results revealed that under adversity stress,HD structure tended to form well-organized structures with large amount of ?-helix.Bioinformatics analysis further implied that HDs from different organisms may protect molecules from damage under stress conditions as the chaperone.3.To test the function of hydrophilic domain from different organisms,we constructed the expression strains containing the whole hydrophilic domains respectively,and examined the survival of these E.coil strains under abiotic stress.As the result,all hydrophilic domain from different organisms can enhance the ability of the hosts to tolerate oxidation and drought resistance.Among them,Cehd has the most significant enhancement,followed by Drhd,then the Bnhd and Ylhd.Additionally,malate dehydrogenase(MDH)and lactate dehydrogenase(LDH),two commonly used enzymes for testing molecular chaperone activity,were used to test the effect of the HD under the condition of vacuum freeze-drying and oxidative stress.The results showed that all hydrophilic domains can protect the activity of MDH and LDH under stress.Cehd displayed the most powerful protection,followed by Drhd,then the Bnhd and Ylhd.Overall,this study demonstrated that the LEA3 hydrophilic domain has the chaperone-like function under stress conditions so that they can protect cells from damage.Notably,the results set the stage for investigating molecular mechanisms of LEA3 protein.