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Prediction To The Key Binding Site Of Pheromone Binding Protein To Cyrtotrachelus Buqueti

Posted on:2020-08-01Degree:MasterType:Thesis
Country:ChinaCandidate:Y L LiuFull Text:PDF
GTID:2393330590498033Subject:Forest Protection
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The olfactory system of insects plays an irreplaceable role in adapting to the environment and has an important influence on their behavior.Cyrtotrachelus buqueti,an important borer forest pest,is harmful to various clumps of bamboo.It's mainly distributed in the south of China and has a great impact on the development of bamboo industry.Pheromone binding proteins(PBPs)are the key substances to control the mating behavior of adults,and their binding mechanism has become a hot research topic.In this paper,the binding mechanism of Cbuq PBP1 was studied,and the key binding sites of Cbuq PBP1 were explored,so as to provide a new method and way to study the mechanism of action of Cbuq PBP1,and lay a foundation for subsequent studies.The 3d structure of Cbuq PBP1 was predicted by homologous modeling,and the Cbuq PBP1 obtained by modeling was molecular docking with the odorants to predict the key sites of protein-odorant binding.The key sites were mutated by site-directed mutation technology and the prokaryotic expression of proprotein Cbuq PBP1 and mutant protein was performed in E.coli.The binding characteristics before and after mutation were compared by fluorescence competitive binding test.The main results are as follows:1.Homology modeling based on amino acid sequences of homologous proteins showed that Cbuq PBP1 possessed the basic structural characteristics of insect pheromone binding proteins.It had six alpha helixes and three disulfide bonds.It was formed by oxidation of six conserved cysteine residues Cys36-Cys67,Cys63-Cys121 and Cys110-Cys130,which played a role in stabilizing the spatial structure of the proteins.The internal binding pocket of Cbuq PBP1 was formed by reverse parallel ? 1,? 3,? 4,? 5,? 6,while ? 2 was located above the pocket,forming a lid-like structure covering the protein binding pocket,which made its structure more stable.There were 10 hydrophobic amino acid residues in the active region of proteins,which participated in the binding of hydrophobic odor molecules and were related to the transport of hydrophobic odor molecules.2.Previous experimental results showed that di(2-ethylhexyl)phthalate and dibutylphthalate had good affinity with Cbuq PBP1.Therefore,on the basis of the three-dimensional structure of the constructed Cbuq PBP1,the protein was docked with the two odorants.According to the binding energy and distance of the docking results,the key binding site of protein-binding pheromone was predicted.By analyzing the docking of Cbuq PBP1 with odor compounds,it was found that Cbuq PBP1 interacted with dibutyl phthalate and di(2-ethylhexyl)phthalate to form CH-pi and hydrogen bonds in Phe-69 and His-53 respectively,so Phe-69 and His-53 were speculated to be the key binding sites of Cbuq PBP1.3.Amino acid substitution was successfully performed for two key binding sites of the predicted Cbuq PBP1 binding pheromone by site-directed mutagenesis using overlapping extended PCR.Histidine(His-53)and phenylalanine(Phe-69)were successfully mutated into alanine(Ala),and then the expression vectors of two mutants and non-mutant genes were transferred to E.coli.After induced by IPT.G(1mM)and cultured at 37 ? for 3 hours,all the proteins were successfully expressed in the supernatant and purified with 85% purity,which provided experimental materials for subsequent experiments.4.Using 1-NPN as fluorescent probe,the competitive binding ability of Cbuq PBP1 and two mutant proteins with di(2-ethylhexyl)phthalate,dibutyl phthalate,cedar and benzothiazole was studied.Phe-69 had an effect on Cbuq PBP1 binding pheromone,but only when the final concentration of cedar and di(2-ethylhexyl phthalate)was greater than50 mM,the fluorescence peak did not decrease to half,which might only affect the conformation of Cbuq PBP1 binding pocket and not the key binding site.After the mutation of His-53,when the final concentration of odor substance was greater than 50 mM,the fluorescence peak did not decrease to half,indicating that the binding ability of Cbuq PBP1 was almost lost,and His-53 was the key binding site of Cbuq PBP1 and was crucial to the function of Cbuq PBP1.In conclusion,the results showed that His-53 of Cbuq PBP1 was the key binding site between protein and odor substance,Phe-69 was the binding site between protein and odor substance,and hydrogen bond was the main binding force between Cbuq PBP1 and odor substance.
Keywords/Search Tags:Cyrtotrachelus buqueti, pheromone binding protein, His-53, fluorescence competitive binding assays
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