Chaperone-like And Catalytic Functions In Deinococcus Radiodurans Of Hydrophilic Protein DohL Involved In Protective Against Oxidative Stress

Previous study unraveled that LEA proteins were functionally associated with organisms'abiotic stress tolerance.According to amino acid sequences and conserved motifs,LEA proteins are classified into seven distinctive groups,and group 3 proteins was well-characterized constitute of conserved motif containing11 amino acids?11-mer?.Nearly all of the LEA3 proteins were identified as intrinsically disordered proteins,which displayed a chaperone-like function protecting protein structure from aggregation.Our previous study discovered three desiccation-associated homologous proteins in D.radiodurans,but their functions were still unclear.In our research,we identified a hydrophilic ordered LEA3 protein,DR1172,which was genus-specific to Deinococcus designating DohL?Deinococcus ordered hydrophilic LEA3,DohL?.Therefore,further investigation of DohL was carried out elaborately to demonstrate its molecular mechanism,from structure to physiological function.The main results obtained were as following;1.Circular dichroism?CD?analysis showed that the second structure of DohL was identified as novel ordered??-helicity is 26.9%?under normal condition,which was basically different with the identified intrinsically-disordered LEA3 protein.Furthermore,the?-helicity of DohL were increased up to 93.4%and 99.8%respectively in the presence of 50%glycerol and 50%trifluoroethanol?TFE?,which tended to be more order and stable.QRT-PCR and digital PCR results shown that dohL expression was reduced under different H₂O₂ concentration,whereas western-blot results shown that DohL protein was largely produced.2.Phenotypic experiments shown that?dohL mutant became more sensitive under oxidation stress compared to the wild-type.Further,gene expression of oxidases?CAT and SOD?and their enzymatic activity were significantly decreased indicating the involvement of DohL to the oxidation resistance of D.radiodurans.3.Size-varied truncated HD proteins can compensate the loss oxidation resistance of?dohL mutant,especially the Com-DHD16?double-time size HD,DHD16,contains 16 motifs?,Com-dohL?whole length DohL,contains 8 conserved motifs?and Com-HD8?only the intact HD8,contains 8 motifs?,which were completely recovering the oxidation resistance.Com-THD4-1?truncated first half HD,THD4-1,contains 4 motifs?and Com-THD4-2?truncated second half HD,THD4-2,4 motifs?bearing the similar and partially-recovery ability.In addition,heterologous expression of these 5 HD derivatives conferred E.coli strains with improved oxidation resistance.Collectively,HD played a crucial role to protect organisms from oxidation and there might be a correlation between the motif numbers with the level of oxidation resistance.4.Protein-protein interaction results shown that both DohL and HDs can interact with LDH respectively and their affinity were conformably increased at the presence of oxidation.Further experiments revealed that additional DohL or HDs can separately protect LDH's enzymatic activity by disaggregation.Accordingly,these DohL and HDs performed a chaperone-like role.5.DohL performed nuclease-like activity to digest the supercoiled?SC?plasmid at the absence of ATP.DohL displayed excellent catalytic activity than T7 endonuclease under oxidation.Therefore,we speculated that DohL has an important function by scavenging DNA fragmentation caused by oxidation.Taken together,unlike canonical identified LEA proteins,DohL was a novel chaperone-like ordered protein,which bears amazingly nuclease activity and assumingly seen as a strategy of D.radiodurans adapting to the extreme environments.