云斑天牛Minus-C OBPs與配基的結(jié)合與釋放機(jī)制
發(fā)布時(shí)間：2018-08-12 08:32
【摘要】：云斑天牛Batocera horsfieldi(Hope),又名云斑白條天牛,在我國(guó)分布廣泛,是林業(yè)上重要的蛀干害蟲(chóng),其靈敏的嗅覺(jué)系統(tǒng)在寄主定位、交配和產(chǎn)卵等方面發(fā)揮著重要的作用。而昆蟲(chóng)的氣味結(jié)合蛋白(OBPs)可以攜帶環(huán)境中的疏水性氣味分子到達(dá)氣味受體,在特異性識(shí)別信息化學(xué)物質(zhì)的過(guò)程中起著重要的功能。若能明確云斑天牛氣味結(jié)合蛋白與配體分子的結(jié)合與釋放機(jī)制,可為其行為調(diào)節(jié)劑的研究提供重要的理論依據(jù)。為此,本課題以云斑天牛為研究對(duì)象,測(cè)定了Bhor OBPs與17種寄主揮發(fā)物的熒光競(jìng)爭(zhēng)結(jié)合特性,并利用計(jì)算機(jī)進(jìn)行同源模建和分子對(duì)接,配合氨基酸定點(diǎn)突變技術(shù),分析了Bhor OBPm2的結(jié)構(gòu)特點(diǎn)及其與配體分子的結(jié)合與釋放機(jī)制。主要研究結(jié)果如下:1.云斑天牛Minus-C OBPs基因克隆、蛋白表達(dá)和純化成功構(gòu)建重組質(zhì)粒p GEX-6P-1-Bhor OBPm1,p ET30a-Bhor OBPm2和p ET17bBhor OBPm4。p GEX-6P-1-Bhor OBPm1在上清中表達(dá),通過(guò)GST 4B親和層析柱獲得Bhor OBPm1的純蛋白;p ET30a-Bhor OBPm2和p ET17b-Bhor OBPm4在包涵體中表達(dá),分別通過(guò)Ni離子親和層析柱及DE52弱陰離子交換柱在體外成功獲得Bhor OBPm2和Bhor OBPm4的純蛋白。2.云斑天牛Minus-C OBPs結(jié)合特性分析以1-NPN為熒光探針?lè)謩e測(cè)定了云斑天牛3個(gè)Minus-C OBPs與寄主揮發(fā)物在酸性和中性環(huán)境下的結(jié)合能力。Bhor OBPm1在中性環(huán)境下與水楊醛有較弱的結(jié)合能力,在酸性環(huán)境下與馬鞭草烯酮有較強(qiáng)結(jié)合能力;Bhor OBPm2在中性環(huán)境與己醛、反-2-己烯醛以及反-2-己烯醇有較強(qiáng)的結(jié)合能力,在酸性條件下僅與反-2-己烯醇、己醛和2-甲基丁醛表現(xiàn)出較弱的結(jié)合能力;Bhor OBPm4在中性環(huán)境下與所有揮發(fā)物都無(wú)結(jié)合能力,在酸性環(huán)境下,僅與檸檬烯氧化物、水楊醛和順-3-己烯醇表現(xiàn)出較弱的結(jié)合能力。3.云斑天牛Bhor OBPm2同源建模與分子對(duì)接以同源蛋白Agam OBP1(2ERB)為模板,建模得到Bhor OBPm2的三維結(jié)構(gòu),其具有六個(gè)α螺旋,兩個(gè)二硫鍵,屬于Minus-C OBPs家族。與模板蛋白Agam OBP1(2ERB)的C端結(jié)構(gòu)相似,Bhor OBPm2的C末端的氨基酸殘基Phe123與Tyr50形成氫鍵,將C端固定在結(jié)合腔的表面。Bhor OBPm2與56種氣味物質(zhì)對(duì)接結(jié)果顯示,六個(gè)α螺旋在Bhor OBPm2的中心形成了一個(gè)疏水性的結(jié)合腔,且在結(jié)合腔的表面形成了兩個(gè)大小不同的開(kāi)口。結(jié)合腔的表面大部分為疏水性區(qū)域,配體小分子主要以疏水作用力固定在腔內(nèi),而C端氨基酸殘基Ile121和Phe123形成了結(jié)合腔表面唯一的極性區(qū)域。4.Bhor OBPm2的氨基酸定點(diǎn)突變及結(jié)合驗(yàn)證熒光競(jìng)爭(zhēng)結(jié)合試驗(yàn)結(jié)果表明,Bhor OBPm2特異性選擇鏈狀配基,且基于碳鏈的長(zhǎng)度進(jìn)行選擇;Bhor OBPm2與配基的結(jié)合能力受p H的影響,中性環(huán)境下的結(jié)合能力明顯強(qiáng)于酸性環(huán)境。將Bhor OBPm2與其他OBPs進(jìn)行比較,我們發(fā)現(xiàn)屬于Minus-C OBPs家族的Bhor OBPm2與屬于Classical OBPs家族的Agam OBP1、Cqui OBP1和Aaeg OBP1具有相似的結(jié)合腔結(jié)構(gòu)以及配基選擇取向,但由于缺失一個(gè)二硫鍵,Bhor OBPm2擁有更大的結(jié)合腔和更廣泛的配基選擇。因此我們猜測(cè)Bhor OBPm2可能是OBPs進(jìn)化過(guò)程中的中間體結(jié)構(gòu)。為探究Bhor OBPm2 C端的作用,我們?cè)O(shè)計(jì)了兩個(gè)Bhor OBPm2的突變體C-ter113和Y50F。通過(guò)對(duì)突變體蛋白的研究發(fā)現(xiàn),Bhor OBPm2的C端在配基釋放的過(guò)程中并非如前人報(bào)道的起到了“蓋子”作用。
[Abstract]:Batocera horsfieldi (Hope), also known as Batocera horsfieldi (Hope), is widely distributed in China and is an important stem-borer in forestry. Its sensitive olfactory system plays an important role in host location, mating and oviposition. The odor-binding proteins (OBPs) of insects can carry hydrophobic odor molecules into the atmosphere. Taste receptors play an important role in the specific recognition of informational substances. If the binding and release mechanism of odor-binding proteins and ligand molecules can be clarified, it will provide an important theoretical basis for the study of behavioral regulators. The fluorescence competitive binding characteristics of the main volatiles were studied by computer homology modeling and molecular docking. The structure characteristics of Bhor OBPm2 and its binding and release mechanism with ligand molecules were analyzed by amino acid site-directed mutagenesis technique. The main results were as follows: 1. Cloning of Minus-C OBPs gene, protein expression and purification were successful. The recombinant plasmids P GEX-6P-1-Bhor OBPm1, P ET30a-Bhor OBPm2 and pET17bhor OBPm4.p GEX-6P-1-Bhor OBPm1 were constructed and expressed in the supernatant. The purified protein of Bhor OBPm1 was obtained by GST 4B affinity chromatography. The expression of P ET30a-Bhor OBPm2 and P ET17b-Bhor OBPm4 in inclusion bodies was detected by Ni ion affinity chromatography and DE52 weak anion exchange column, respectively. Bhor OBPm 2 and Bhor OBPm4 were successfully obtained in vitro. 2. The binding properties of three Minus-C OBPs from A. yunnanensis to host volatiles in acidic and neutral environments were determined by using 1-NPN as fluorescent probe. Bhor OBPm1 had weak binding ability to salicylaldehyde in neutral environments, and in acidic and neutral environments. Bhor OBPm2 has strong binding ability with hexanal, trans-2-hexenal and trans-2-hexenol in neutral environment, but weak binding ability with trans-2-hexenol, hexanal and 2-methylbutyraldehyde in acidic environment; Bhor OBPm4 has no binding ability with all volatiles in neutral environment. The binding ability of Bhor OBPm2 was weaker than that of citrone oxides, salicylaldehyde and cis-3-hexenol in acidic environment. 3. Homologous modeling and molecular docking of Bhor OBPm2 were performed using homologous protein Agam OBP1 (2ERB) as template, and the three-dimensional structure of Bhor OBPm2 was modeled. It has six alpha helixes and two disulfide bonds, belonging to Minus-C OBPs. Similar to the C-terminal structure of the template protein Agam OBP1 (2ERB), the amino acid residue Phe123 at the C-terminal of Bhor OBPm2 forms a hydrogen bond with Tyr50 and fixes the C-terminal on the surface of the binding cavity. The docking results of Bhor OBPm2 with 56 odor compounds show that six alpha-helixes form a hydrophobic binding cavity in the center of Bhor OBPm2 and are in the binding cavity. The surface of the binding cavity is mostly hydrophobic, and the small ligand molecules are mainly fixed in the cavity by hydrophobic force. The C-terminal amino acid residues Ile121 and Phe123 form the unique polar region on the surface of the binding cavity. 4. Site-directed mutagenesis of amino acids in Bhor OBPm2 and binding verification fluorescence competitive binding test The results show that Bhor OBPm2 selectively chooses chain-like ligands based on the length of carbon chain, and the binding ability of Bhor OBPm2 to ligands is affected by P H. The binding ability of Bhor OBPm2 in neutral environment is obviously stronger than that in acidic environment. Gam OBP1, Cqui OBP1 and Aaeg OBP1 of the cal OBPs family have similar binding cavity structures and ligand selection orientations, but Bhor OBPm2 has larger binding cavity and wider ligand selection due to the absence of a disulfide bond. Therefore, we speculate that Bhor OBPm2 may be an intermediate structure in the evolution of OBPs. Two mutants of Bhor OBPm2, C-ter113 and Y50F, were designed. It was found that the C-terminus of Bhor OBPm2 did not act as a "cap" during the release of the ligand.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S763.38
本文編號(hào)：2178511
[Abstract]:Batocera horsfieldi (Hope), also known as Batocera horsfieldi (Hope), is widely distributed in China and is an important stem-borer in forestry. Its sensitive olfactory system plays an important role in host location, mating and oviposition. The odor-binding proteins (OBPs) of insects can carry hydrophobic odor molecules into the atmosphere. Taste receptors play an important role in the specific recognition of informational substances. If the binding and release mechanism of odor-binding proteins and ligand molecules can be clarified, it will provide an important theoretical basis for the study of behavioral regulators. The fluorescence competitive binding characteristics of the main volatiles were studied by computer homology modeling and molecular docking. The structure characteristics of Bhor OBPm2 and its binding and release mechanism with ligand molecules were analyzed by amino acid site-directed mutagenesis technique. The main results were as follows: 1. Cloning of Minus-C OBPs gene, protein expression and purification were successful. The recombinant plasmids P GEX-6P-1-Bhor OBPm1, P ET30a-Bhor OBPm2 and pET17bhor OBPm4.p GEX-6P-1-Bhor OBPm1 were constructed and expressed in the supernatant. The purified protein of Bhor OBPm1 was obtained by GST 4B affinity chromatography. The expression of P ET30a-Bhor OBPm2 and P ET17b-Bhor OBPm4 in inclusion bodies was detected by Ni ion affinity chromatography and DE52 weak anion exchange column, respectively. Bhor OBPm 2 and Bhor OBPm4 were successfully obtained in vitro. 2. The binding properties of three Minus-C OBPs from A. yunnanensis to host volatiles in acidic and neutral environments were determined by using 1-NPN as fluorescent probe. Bhor OBPm1 had weak binding ability to salicylaldehyde in neutral environments, and in acidic and neutral environments. Bhor OBPm2 has strong binding ability with hexanal, trans-2-hexenal and trans-2-hexenol in neutral environment, but weak binding ability with trans-2-hexenol, hexanal and 2-methylbutyraldehyde in acidic environment; Bhor OBPm4 has no binding ability with all volatiles in neutral environment. The binding ability of Bhor OBPm2 was weaker than that of citrone oxides, salicylaldehyde and cis-3-hexenol in acidic environment. 3. Homologous modeling and molecular docking of Bhor OBPm2 were performed using homologous protein Agam OBP1 (2ERB) as template, and the three-dimensional structure of Bhor OBPm2 was modeled. It has six alpha helixes and two disulfide bonds, belonging to Minus-C OBPs. Similar to the C-terminal structure of the template protein Agam OBP1 (2ERB), the amino acid residue Phe123 at the C-terminal of Bhor OBPm2 forms a hydrogen bond with Tyr50 and fixes the C-terminal on the surface of the binding cavity. The docking results of Bhor OBPm2 with 56 odor compounds show that six alpha-helixes form a hydrophobic binding cavity in the center of Bhor OBPm2 and are in the binding cavity. The surface of the binding cavity is mostly hydrophobic, and the small ligand molecules are mainly fixed in the cavity by hydrophobic force. The C-terminal amino acid residues Ile121 and Phe123 form the unique polar region on the surface of the binding cavity. 4. Site-directed mutagenesis of amino acids in Bhor OBPm2 and binding verification fluorescence competitive binding test The results show that Bhor OBPm2 selectively chooses chain-like ligands based on the length of carbon chain, and the binding ability of Bhor OBPm2 to ligands is affected by P H. The binding ability of Bhor OBPm2 in neutral environment is obviously stronger than that in acidic environment. Gam OBP1, Cqui OBP1 and Aaeg OBP1 of the cal OBPs family have similar binding cavity structures and ligand selection orientations, but Bhor OBPm2 has larger binding cavity and wider ligand selection due to the absence of a disulfide bond. Therefore, we speculate that Bhor OBPm2 may be an intermediate structure in the evolution of OBPs. Two mutants of Bhor OBPm2, C-ter113 and Y50F, were designed. It was found that the C-terminus of Bhor OBPm2 did not act as a "cap" during the release of the ligand.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S763.38
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