Effects Of Ordered Structure On Stress Tolerance Of Chaperone-Like Protein Dohl In Deinococcus Radiodurans

Deinococcus radiodurans is extremely resistant to oxidation,freezing and other abiotic stresses.Lots of studies have shown that the chaperone-like proteins LEA3 with random coil as secondary structure were involved in the stress resistance process of this strain.Previous studies have shown that the chaperone-like protein DohL in Deinococcus radiodurans belonged to the LEA3 protein family and was characterized by 8 conserved 11-mer amino acids motifs,which was consistent with the characteristics of the LEA3 protein family.But unlike reported LEA3 proteins with random coil as secondary structure,DohL proteins can form intrinsically ordered secondary structure,which mainly include?-helix.The folded order of this structure may be related to stress tolerance of DohL and the adaptability to extreme environments of Deinococcus radiodurans.In this study,the order of DohL protein was reduced by site-directed mutation,and structure-function relationships of DohL protein was clarified by circular dichroism,constructing recombinant E.coli strains and complementary strains.The main results obtained are as follows:1.Bioinfoimatic analysis showed that the N-terminal sequence?at position 1-103?of the DohL protein was conserved in the Deinococcus genus and this sequence may be the key region for the formation of ordered secondary structure of DohL.The results of the analysis showed that Leucine?L?,Tryptophan?W?and Isoleucine?I?could promote order and were highly conserved.Therefore,the W at position 61 and I at position 75 were mutated to Proline?P?,and the mutant protein was named as WIP,and consecutive L?leucine?at position 14 to 17,W at position 61 and I at position 75 were mutated to P,and this mutant protein was named as LWIP.The order of prediction by software of the two proteins after mutation is reduced.Compared with the DohL protein,the folded order of this 3 proteins decreased along this order:DohL>WIP>LWIP.Further,the circular dichroism was carried on to determine the order of secondary structure of DohL and mutant proteins.The results showed that the folded order of the 3proteins was DohL:82.5%,WIP:62.2%,LWIP:51.4%,which was consistent with the prediction.2.The constructed recombinant E.coli were used for heterologous expression and phenotype experiments.Under the conditions of oxidation and freezing stress,the survivability of recombinant strains expressing mutant proteins were weaker than that of recombinant strain expressing DohL protein.In order to study the ability of DohL and two mutant proteins to protect LDH enzyme under freezing-thawing and H₂O₂ conditions,we expressed and purified DohL,WIP and LWIP proteins.The enzyme activity tested in vitro indicated that the 3 proteins could protect the activity of lactate dehydrogenase?LDH?from stresses,and they had chaperone-like function.The protective power decreased along this order:DohL>WIP>LWIP,which indicated that protective function of DohL was weakened with decreased order.3.In order to study the function of DohL,WIP and LWIP,we constructed Com-dohl,Com-wip and Com-lwip complementary strains,and stress-tolerance experiment such as high-concentration H₂O₂among the wild-type?DR?,mutant??dohl?and complementary strains was carried on for further study.The survival experiment showed that the complementary strains Com-dohl,Com-wip and Com-lwip could compensate the lost phenotype of mutant?dohl to extent under oxidant stress,and the phenotype-recover ability of 3 complementary strains decreased along this order:Com-dohl>Com-wip>Com-lwip,which indicated that stress tolerance of DohL was weakened with decreased order,leading to reducing oxidative resistance of the strain.Furthermore,the total antioxidant capacity of the wild type,the mutant strain and the complementary strains was determined under the 80 mM H₂O₂ condition.The results showed that the total antioxidant activity of the complementary strains Com-wip and Com-lwip was weaker than that of the complementary strain Com-dohl,which indicated that the DohL protein with reduced order maybe affect resistant function,making attenuation with the oxidative stress resistance of strain,leading to speculation that the amino acids at highly conserved sites play an important role in the stress tolerance of the DohL protein.Taken together,the N-terminal region of the DohL protein is the key region for the ordered folding of this protein,and the stress tolerance of the DohL protein is decreased with reduced order.The results indicate that the the composition and proportion of amino acids in Deinococcus radiodurans contribute to the correct folding and stress tolerance of DohL protein,which may be the result of adapting the strain to extreme environments.