本文主要研究内容
作者刘野（2019）在《基于分子动力学模拟研究蛋白质二级结构变化对其活性的影响》一文中研究指出：蛋白质作为生命体的基础物质,参与到生命活动的各个环节。由多个二级结构组成的蛋白质三级结构决定蛋白质的结构与功能,蛋白质二级结构的改变往往会影响蛋白质的活性。传统的分子生物学实验技术无法从微观上检测二级结构的变化,也无法分析二级结构变化调控蛋白质活性的分子机理。分子动力学模拟是随着生物科学技术与计算机科学技术的发展,应运而生的一种新兴交叉的生物学手段,被应用于生物大分子催化机制、蛋白构象改变、酶与配体相互作用机制的研究中,发挥了其独特的优势,在很多方面成为实验难以替代的方法。本论文以人MEK1、玉米赤烯酮降解酶、嗜热菌Argonaute和环状芽孢杆菌的转氨酶为蛋白质分子模型,采用多种计算生物学的方法(分子动力学模拟、蛋白质网络分析、结合自由能计算、分子对接),针对几种重要蛋白进行深入的研究,对蛋白质二级结构调控蛋白质活性的分子机理进行了深入的讨论。论文的具体研究内容如下:1.MEK1二级结构变化影响其催化活性的分子机制大约30%的癌症发生与MAPK信号转导通路的激活有关,丝裂原活化蛋白激酶激酶(MEK1)是该条信号通路的关键组成部分。据报道,在很多类型的癌症中,MEK1分子氨基酸的突变能够增强其活性,从而导致癌细胞的增殖和分化。但是突变引起蛋白构象变化以及调控其活性的分子机制并不清楚。本研究对野生型MEK1(无活性)、一种无活性突变(A52V)、五种有活性突变(K57N、P124S、E203K、I103和E102_I103缺失突变)以及磷酸化的MEK1(有活性)进行了分子动力学模拟,和蛋白质网络以及MM-PBSA的分析。蛋白质结构网络和氢键分析表明,位于C Helix(Residue 105-121)附近的残基发生突变,降低了C Helix的稳定性,进而破坏了MEK1中Activation segment(Residue 216-222)和C Helix之间的相互作用,并伴随着Activation segment的二级结构由Helix变成Coil,最终导致Activation segment远离MEK1蛋白的主体,在蛋白表面形成一个开放的缺口,即蛋白由Close状态转变成Open状态。氢键分析和MM-PBSA计算结果说明有活性突变能够通过降低MEK1和抑制剂ANP之间的结合亲和力以抵消抑制剂的抑制作用。这些理论计算的结果都能充分地阐释有活性突变增强MEK1活性的分子机制,为二级结构变化影响蛋白活性分子机制的研究提供了理论依据。2.玉米赤烯酮降解酶二级结构变化影响其对α-ZOL降解效率的分子机制玉米赤烯酮(ZEN)及其衍生物是从发霉谷类中提取出的污染物,因其污染程度大、难降解的性质,给农业和工业经济带来很大的损失。国内外学者一直致力于寻找高效的降解该污染物的方法。玉米赤烯酮降解酶(ZHD)是一种典型的α/β水解酶,也是唯一能够降解玉米赤烯酮的酶,但是野生型ZHD对于玉米赤烯酮衍生物的降解效率很低。最近的研究表明,V153H突变能特异性的增强ZHD对α-ZOL的降解效率。为了分析V153H突变提高ZHD对α-ZOL的催化效率的原因,采用Gromacs软件对野生型/α-ZOL、野生型/β-ZOL、V153H/α-ZOL和V153H/β-ZOL四个复合体进行了分子动力学模拟以及蛋白质网络分析。理论计算结果显示,V153H突变导致了位于ZHD中Cap domain(Residue Gly161-Thr190)的Helix消失,从而增强了Cap domain和Residue Met241-Tyr245之间的联系和氢键相互作用,影响了催化残基His242的相对位置。α-ZOL和ZHD的结合促使α-ZOL中的OAE原子与His242的NE2原子的距离增大,为His242侧链进入催化活性部位提供了足够的空间,从而提高了ZHD对α-ZOL的催化活性。此外,α-ZOL更容易和V153H突变体形成合适的催化进攻角度和距离,以保证催化反应的进行。丙氨酸扫描结果表明,位于Cap domain区域残基的突变能够明显改变ZHD与α-ZOL和β-ZOL之间的亲和力。我们的模拟结果为ZHD催化特异性机理的研究提供了有用的理论依据。3.嗜热菌Argonaute蛋白二级结构变化影响其催化活性的分子机制来自于嗜热菌Thermus thermophiles的Argonaute(Tt Ago)蛋白因其能特异性切割外源核酸的能力,成为国内外学者研究的焦点。但因为Argonaute蛋白需要在高温条件下才具有活性的特点,很大程度上限制了它在生物学领域中的应用。因此,我们在310、324和338K条件下对Tt Ago/DNA复合物进行分子动力学模拟和结合自由能的计算,探索高温维持Tt Ago活性的分子机制,为改造出低温的Argonaute酶提供理论支持。模拟结果表明,在低温条件下,位于Residue Glu507-Arg513处的β-折叠消失,导致残基Glu512远离催化残基Asp546和Asp478,从而导致催化活性的下降,在高温条件的模拟中未观察到这种现象。对蛋白通道的分析结果显示,高温可使Tt Ago蛋白的核酸结合通道变宽,有利于Guide DNA序列和Target DNA序列的进入和离去。除此之外,Tt Ago和DNA的相互作用在324和338K的条件下更加稳定。高温还能在很大程度上降低与DNA结合表面的溶剂可及性表面积,从而影响与DNA的亲水性相互作用。我们的模拟研究揭示了温度通过调控嗜热菌Tt Ago二级结构从而调控其活性的分子机制,为其在基因编辑领域中的应用提供了一定程度的理论支持。4.基于多重分子动力学模拟研究转氨酶Btr R的催化选择特异性井冈霉烯胺(valienamine)是一种带有侧链的不饱和环醇假氨基糖类物质,根据手性碳原子的特异性,存在valienamine和β-valienamine两种同分异构体。因为β-valienamine的衍生物是治疗溶酶体贮存性疾病有效的化学伴侣,其合成路线愈来愈受到人们的关注。有报道称来自于环状芽孢杆菌的转氨酶Btr R在辅酶PLP的帮助下,可以将valienone特异性催化成β-valienamine,且纯度在99%以上。为了研究来自于环状芽孢杆菌的转氨酶Btr R对产物井冈霉烯胺(valienamine)的立体选择性机理,分别对valienamine/PLP/Btr R和β-valienamine/PLP/Btr R两个复合体进行了常规分子动力学模拟和拉伸分子动力学模拟。理论计算结果显示,当β-valienamine存在时,会通过调节Btr R的二级结构使得辅酶PLP和Btr R之间氢键几率增加以及结合自由能的升高,有助于催化反应的发生。β-valienamine能够维持Btr R分子内碱性氨基酸和芳香族氨基酸之间形成阳离子共轭,从而增强Btr R的稳定性及催化活性。拉伸分子动力学模拟结果显示,valienamine在解离过程中能和通道上很多的氨基酸形成相互作用,导致其需要更大的外力才能从Btr R蛋白中解离,这说明β-valienamine更容易从转氨酶Btr R的通道中解离出来,从而为下一轮底物的结合提供空间。所有计算结果表明无论是从催化反应发生的角度,还是从产物离去的角度,Btr R都更倾向于催化底物生成β-valienamine,即对β-valienamine具有极高的选择特异性。
Abstract
dan bai zhi zuo wei sheng ming ti de ji chu wu zhi ,can yu dao sheng ming huo dong de ge ge huan jie 。you duo ge er ji jie gou zu cheng de dan bai zhi san ji jie gou jue ding dan bai zhi de jie gou yu gong neng ,dan bai zhi er ji jie gou de gai bian wang wang hui ying xiang dan bai zhi de huo xing 。chuan tong de fen zi sheng wu xue shi yan ji shu mo fa cong wei guan shang jian ce er ji jie gou de bian hua ,ye mo fa fen xi er ji jie gou bian hua diao kong dan bai zhi huo xing de fen zi ji li 。fen zi dong li xue mo ni shi sui zhao sheng wu ke xue ji shu yu ji suan ji ke xue ji shu de fa zhan ,ying yun er sheng de yi chong xin xing jiao cha de sheng wu xue shou duan ,bei ying yong yu sheng wu da fen zi cui hua ji zhi 、dan bai gou xiang gai bian 、mei yu pei ti xiang hu zuo yong ji zhi de yan jiu zhong ,fa hui le ji du te de you shi ,zai hen duo fang mian cheng wei shi yan nan yi ti dai de fang fa 。ben lun wen yi ren MEK1、yu mi chi xi tong jiang jie mei 、shi re jun Argonautehe huan zhuang ya bao gan jun de zhuai an mei wei dan bai zhi fen zi mo xing ,cai yong duo chong ji suan sheng wu xue de fang fa (fen zi dong li xue mo ni 、dan bai zhi wang lao fen xi 、jie ge zi you neng ji suan 、fen zi dui jie ),zhen dui ji chong chong yao dan bai jin hang shen ru de yan jiu ,dui dan bai zhi er ji jie gou diao kong dan bai zhi huo xing de fen zi ji li jin hang le shen ru de tao lun 。lun wen de ju ti yan jiu nei rong ru xia :1.MEK1er ji jie gou bian hua ying xiang ji cui hua huo xing de fen zi ji zhi da yao 30%de ai zheng fa sheng yu MAPKxin hao zhuai dao tong lu de ji huo you guan ,si lie yuan huo hua dan bai ji mei ji mei (MEK1)shi gai tiao xin hao tong lu de guan jian zu cheng bu fen 。ju bao dao ,zai hen duo lei xing de ai zheng zhong ,MEK1fen zi an ji suan de tu bian neng gou zeng jiang ji huo xing ,cong er dao zhi ai xi bao de zeng shi he fen hua 。dan shi tu bian yin qi dan bai gou xiang bian hua yi ji diao kong ji huo xing de fen zi ji zhi bing bu qing chu 。ben yan jiu dui ye sheng xing MEK1(mo huo xing )、yi chong mo huo xing tu bian (A52V)、wu chong you huo xing tu bian (K57N、P124S、E203K、I103he E102_I103que shi tu bian )yi ji lin suan hua de MEK1(you huo xing )jin hang le fen zi dong li xue mo ni ,he dan bai zhi wang lao yi ji MM-PBSAde fen xi 。dan bai zhi jie gou wang lao he qing jian fen xi biao ming ,wei yu C Helix(Residue 105-121)fu jin de can ji fa sheng tu bian ,jiang di le C Helixde wen ding xing ,jin er po huai le MEK1zhong Activation segment(Residue 216-222)he C Helixzhi jian de xiang hu zuo yong ,bing ban sui zhao Activation segmentde er ji jie gou you Helixbian cheng Coil,zui zhong dao zhi Activation segmentyuan li MEK1dan bai de zhu ti ,zai dan bai biao mian xing cheng yi ge kai fang de que kou ,ji dan bai you Closezhuang tai zhuai bian cheng Openzhuang tai 。qing jian fen xi he MM-PBSAji suan jie guo shui ming you huo xing tu bian neng gou tong guo jiang di MEK1he yi zhi ji ANPzhi jian de jie ge qin he li yi di xiao yi zhi ji de yi zhi zuo yong 。zhe xie li lun ji suan de jie guo dou neng chong fen de chan shi you huo xing tu bian zeng jiang MEK1huo xing de fen zi ji zhi ,wei er ji jie gou bian hua ying xiang dan bai huo xing fen zi ji zhi de yan jiu di gong le li lun yi ju 。2.yu mi chi xi tong jiang jie mei er ji jie gou bian hua ying xiang ji dui α-ZOLjiang jie xiao lv de fen zi ji zhi yu mi chi xi tong (ZEN)ji ji yan sheng wu shi cong fa mei gu lei zhong di qu chu de wu ran wu ,yin ji wu ran cheng du da 、nan jiang jie de xing zhi ,gei nong ye he gong ye jing ji dai lai hen da de sun shi 。guo nei wai xue zhe yi zhi zhi li yu xun zhao gao xiao de jiang jie gai wu ran wu de fang fa 。yu mi chi xi tong jiang jie mei (ZHD)shi yi chong dian xing de α/βshui jie mei ,ye shi wei yi neng gou jiang jie yu mi chi xi tong de mei ,dan shi ye sheng xing ZHDdui yu yu mi chi xi tong yan sheng wu de jiang jie xiao lv hen di 。zui jin de yan jiu biao ming ,V153Htu bian neng te yi xing de zeng jiang ZHDdui α-ZOLde jiang jie xiao lv 。wei le fen xi V153Htu bian di gao ZHDdui α-ZOLde cui hua xiao lv de yuan yin ,cai yong Gromacsruan jian dui ye sheng xing /α-ZOL、ye sheng xing /β-ZOL、V153H/α-ZOLhe V153H/β-ZOLsi ge fu ge ti jin hang le fen zi dong li xue mo ni yi ji dan bai zhi wang lao fen xi 。li lun ji suan jie guo xian shi ,V153Htu bian dao zhi le wei yu ZHDzhong Cap domain(Residue Gly161-Thr190)de Helixxiao shi ,cong er zeng jiang le Cap domainhe Residue Met241-Tyr245zhi jian de lian ji he qing jian xiang hu zuo yong ,ying xiang le cui hua can ji His242de xiang dui wei zhi 。α-ZOLhe ZHDde jie ge cu shi α-ZOLzhong de OAEyuan zi yu His242de NE2yuan zi de ju li zeng da ,wei His242ce lian jin ru cui hua huo xing bu wei di gong le zu gou de kong jian ,cong er di gao le ZHDdui α-ZOLde cui hua huo xing 。ci wai ,α-ZOLgeng rong yi he V153Htu bian ti xing cheng ge kuo de cui hua jin gong jiao du he ju li ,yi bao zheng cui hua fan ying de jin hang 。bing an suan sao miao jie guo biao ming ,wei yu Cap domainou yu can ji de tu bian neng gou ming xian gai bian ZHDyu α-ZOLhe β-ZOLzhi jian de qin he li 。wo men de mo ni jie guo wei ZHDcui hua te yi xing ji li de yan jiu di gong le you yong de li lun yi ju 。3.shi re jun Argonautedan bai er ji jie gou bian hua ying xiang ji cui hua huo xing de fen zi ji zhi lai zi yu shi re jun Thermus thermophilesde Argonaute(Tt Ago)dan bai yin ji neng te yi xing qie ge wai yuan he suan de neng li ,cheng wei guo nei wai xue zhe yan jiu de jiao dian 。dan yin wei Argonautedan bai xu yao zai gao wen tiao jian xia cai ju you huo xing de te dian ,hen da cheng du shang xian zhi le ta zai sheng wu xue ling yu zhong de ying yong 。yin ci ,wo men zai 310、324he 338Ktiao jian xia dui Tt Ago/DNAfu ge wu jin hang fen zi dong li xue mo ni he jie ge zi you neng de ji suan ,tan suo gao wen wei chi Tt Agohuo xing de fen zi ji zhi ,wei gai zao chu di wen de Argonautemei di gong li lun zhi chi 。mo ni jie guo biao ming ,zai di wen tiao jian xia ,wei yu Residue Glu507-Arg513chu de β-she die xiao shi ,dao zhi can ji Glu512yuan li cui hua can ji Asp546he Asp478,cong er dao zhi cui hua huo xing de xia jiang ,zai gao wen tiao jian de mo ni zhong wei guan cha dao zhe chong xian xiang 。dui dan bai tong dao de fen xi jie guo xian shi ,gao wen ke shi Tt Agodan bai de he suan jie ge tong dao bian kuan ,you li yu Guide DNAxu lie he Target DNAxu lie de jin ru he li qu 。chu ci zhi wai ,Tt Agohe DNAde xiang hu zuo yong zai 324he 338Kde tiao jian xia geng jia wen ding 。gao wen hai neng zai hen da cheng du shang jiang di yu DNAjie ge biao mian de rong ji ke ji xing biao mian ji ,cong er ying xiang yu DNAde qin shui xing xiang hu zuo yong 。wo men de mo ni yan jiu jie shi le wen du tong guo diao kong shi re jun Tt Agoer ji jie gou cong er diao kong ji huo xing de fen zi ji zhi ,wei ji zai ji yin bian ji ling yu zhong de ying yong di gong le yi ding cheng du de li lun zhi chi 。4.ji yu duo chong fen zi dong li xue mo ni yan jiu zhuai an mei Btr Rde cui hua shua ze te yi xing jing gang mei xi an (valienamine)shi yi chong dai you ce lian de bu bao he huan chun jia an ji tang lei wu zhi ,gen ju shou xing tan yuan zi de te yi xing ,cun zai valienaminehe β-valienamineliang chong tong fen yi gou ti 。yin wei β-valienaminede yan sheng wu shi zhi liao rong mei ti zhu cun xing ji bing you xiao de hua xue ban lv ,ji ge cheng lu xian yu lai yu shou dao ren men de guan zhu 。you bao dao chen lai zi yu huan zhuang ya bao gan jun de zhuai an mei Btr Rzai fu mei PLPde bang zhu xia ,ke yi jiang valienonete yi xing cui hua cheng β-valienamine,ju chun du zai 99%yi shang 。wei le yan jiu lai zi yu huan zhuang ya bao gan jun de zhuai an mei Btr Rdui chan wu jing gang mei xi an (valienamine)de li ti shua ze xing ji li ,fen bie dui valienamine/PLP/Btr Rhe β-valienamine/PLP/Btr Rliang ge fu ge ti jin hang le chang gui fen zi dong li xue mo ni he la shen fen zi dong li xue mo ni 。li lun ji suan jie guo xian shi ,dang β-valienaminecun zai shi ,hui tong guo diao jie Btr Rde er ji jie gou shi de fu mei PLPhe Btr Rzhi jian qing jian ji lv zeng jia yi ji jie ge zi you neng de sheng gao ,you zhu yu cui hua fan ying de fa sheng 。β-valienamineneng gou wei chi Btr Rfen zi nei jian xing an ji suan he fang xiang zu an ji suan zhi jian xing cheng yang li zi gong e ,cong er zeng jiang Btr Rde wen ding xing ji cui hua huo xing 。la shen fen zi dong li xue mo ni jie guo xian shi ,valienaminezai jie li guo cheng zhong neng he tong dao shang hen duo de an ji suan xing cheng xiang hu zuo yong ,dao zhi ji xu yao geng da de wai li cai neng cong Btr Rdan bai zhong jie li ,zhe shui ming β-valienaminegeng rong yi cong zhuai an mei Btr Rde tong dao zhong jie li chu lai ,cong er wei xia yi lun de wu de jie ge di gong kong jian 。suo you ji suan jie guo biao ming mo lun shi cong cui hua fan ying fa sheng de jiao du ,hai shi cong chan wu li qu de jiao du ,Btr Rdou geng qing xiang yu cui hua de wu sheng cheng β-valienamine,ji dui β-valienamineju you ji gao de shua ze te yi xing 。
论文参考文献
论文详细介绍
论文作者分别是来自吉林大学的刘野，发表于刊物吉林大学2019-06-25论文，是一篇关于丝裂原活化蛋白激酶激酶论文,玉米赤烯酮降解酶论文,蛋白论文,转氨酶论文,分子模拟论文,蛋白网络分析论文,结合自由能计算论文,分子对接论文,通道分析论文,蛋白质二级结构变化论文,吉林大学2019-06-25论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自吉林大学2019-06-25论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
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