肌质网钙泵作用机理的相关问题研究

发布时间：2018-01-07 19:18

  本文关键词：肌质网钙泵作用机理的相关问题研究　出处：《山东大学》2008年博士论文　论文类型：学位论文

  更多相关文章： 肌质网钙泵 钙通道 耦合作用模式 环境因素影响 离子选择性 钙离子跨膜传输机理

【摘要】： 骨骼肌内质网中的钙离子跨膜传输是一个通过钙泵和钙离子通道的形式传输钙离子的过程,它在控制肌肉收缩和生热方面都有非常重要的作用,其活性的大小是各种细胞能量代谢及功能有无损伤的重要指标。人体内钙泵的失调可以导致多种疾病的发生,但现在对于钙泵和钙通道的作用机制尚无明确的认识。此外,钙离子的跨膜传输过程还有待于进一步的考察。本论文围绕着生物体中钙离子跨膜传输过程中所涉及的与肌质网钙泵和钙通道有关的功能区域分子构象的变化、能量传递、钙离子与各活性位点的耦合作用以及离子选择性等有关问题,用理论化学方法深入研究了钙泵和钙通道的功能原理和本质。同时,结合构效分析及分子间各类强弱相互作用的研究,探讨了氨基酸残基、溶剂分子等环境因素和结构因素等对钙离子传输机制的影响和调控作用,并取得了一些有意义的研究成果,为认识离子泵和离子通道提供了重要的理论信息。主要的研究成果与创新简述如下: 酸性氨基酸与钙离子的耦合特征及其生物意义Ca~(2+)-ATP酶中的酸性氨基酸在Ca~(2+)跨膜传输过程中有重要的作用,它们既可以作为耦合配体直接参与反应,又可作为探测Ca~(2+)结合与否的“探测器”。因此,在本论文中,我们首先系统考察了具有酸性边链的谷氨酸分子和Ca~(2+)的相互作用及其生物意义。密度泛函理论的研究结果表明,在气相中,存在16种稳定的谷氨酸-Ca~(2+)异构体,分别对应着谷氨酸配体和Ca~(2+)的三齿、双齿和单齿耦合的构型特征。其中最稳定的构象对应着Ca~(2+)与三个氧原子(分别为边链的两个羧基氧和一个主链羰基氧)相互作用的模式,并且在结合Ca~(2+)的同时伴随着一个质子转移的现象。次稳定的构象也对应着一个三齿耦合的形式(Ca~(2+)与两个羰基氧和一个氨基氮相互作用),它的能量仅比最稳定的复合物高2.1 kcal/mol。所有复合物的构型和能量特征都从耦合形式、相对能、静电作用、形变能、电荷布局分析、轨道键合特征和分子内氢键等方面进行了研究。结果表明,谷氨酸的三个活性官能团与Ca~(2+)的结合能力如下:羰基氧＞氨基氮＞羟基氧。另外,谷氨酸总是以尽可能多的活性基团与Ca~(2+)进行耦合。由于构型和能量上的相似性,不同复合物之间的相互转化也极易发生。实际生命过程中的肌质网Ca~(2+)跨膜传导细节可以用我们的计算结果解释如下:当Ca~(2+)-ATPase处于E1构型,即Ca~(2+)结合在高亲和位点时,谷氨酸会与Ca~(2+)以较大的强度进行耦合,此时对应的复合物形式为三齿或双齿耦合;当Ca~(2+)-ATP酶转化到具有低亲和位点的E2构型时,谷氨酸-Ca~(2+)会以不稳定的形式(如单齿耦合模式)存在,因为此种耦合形式有利于Ca~(2+)从结合位释放到内腔中。此外,频率分析表明,在复合物中,C=O伸缩振动模式具有最大强度的红外吸收峰,并且在结合了Ca~(2+)之后,这些峰都会红移。 谷氨酸-钙离子复合物的逐步水合效应及其生物意义在实际的生命体中,反应都是在溶液或蛋白环境下进行的。而在溶剂分子的作用下,金属离子-配体的作用模式会与气态中有所区别。为了更好地接近于真实的生命过程,我们又采用密度泛函理论(DFT)研究了逐步水合对谷氨酸-Ca~(2+)体系的影响。研究内容包括水合反应的热力学参数,逐步水合能及精确的构型信息等。同时,为了进一步考察水合作用的影响,本文也对相关的电荷转移、键合分析以及红外光谱特征进行了研究。计算结果表明,在静电作用、电荷转移、电子轨道效应和离子-配体、配体-配体排斥力的共同影响下,水分子的增加削弱了Ca-O键的强度,并导致了(C-)O-Ca-O(-C)键角的减小。而当水分子同时结合在Ca~(2+)的第一耦合层和第二耦合层时,外围的水合使得内层水分子靠近Ca~(2+),而内层的羧基氧逐渐远离Ca~(2+)。此外,由于氢键作用的增加,逐步水合过程可以使谷氨酸-Ca~(2+)体系变得更稳定。所有的逐步水合反应均为焓驱动过程,但是当耦合的水分子位于第一层时,这种逐步水合过程并不能无限制地进行下去,而是有一个最大的水合数。本文的研究数据表明此数值为6或7。在蛋白质数据库(PDB)中,Ca~(2+)的平均耦合数也为6,而在可溶性的生物体钙蛋白中,最常见的耦合数为7,与我们的计算结果相一致。同时,溶剂化模型计算(SCRF)和高水平的MP2计算也证实了计算结果的准确性。在计算过程中,还发现了一个非常重要的配体交换的现象,即:无论水分子耦合在Ca~(2+)的第一层还是第二层,随着耦合数的增加,它们都会逐步削弱谷氨酸和Ca~(2+)的作用强度,直至其中的一个耦合水分子代替谷氨酸配体的位置,使谷氨酸由内层耦合模式转变为外层耦合模式。在真实的肌质网钙通道中,Ca~(2+)处于蛋白质空穴中,为氨基酸残基所包围:而当其经由Ca~(2+)释放通道进入内腔时,内腔中的水分子会部分或全部代替原来的氨基酸残基。此过程与我们的模拟计算过程是及其类似的,因此我们的研究完全可以提供相关的理论数据来加深人们对于Ca~(2+)通道作用的理解。另外,我们还对所采用的计算模型与实际生物体中肌质网钙通道的相似性和不同点进行了比较研究。 离子选择性的本质及其影响因素在Mg~(2+)浓度比Ca~(2+)浓度高数千倍的情况下,肌质网Ca~(2+)-ATPase仍可以特异性地选择Ca~(2+)进行跨膜传导。离子选择性是Ca~(2+)-ATPase的一个重要特征。我们所进行的密度泛函的计算结果表明,无论是由脂肪族还是芳香族氨基酸组成的蛋白空穴都更容易结合Mg~(2+)而非Ca~(2+),这主要是由Mg~(2+)和配体之间更强的静电相互作用引起的。进一步的研究表明,氨基酸对于水合阳离子的亲合力受金属离子的特性、氨基酸配体的电负性和结合模式等因素的影响。相对于Ca~(2+)来说,Mg~(2+)有更小的半径和更大的电荷密度。因此,如果结合到同一个配体上,Mg~(2+)会表现出比Ca~(2+)更强的静电相互作用。另一方面,当配体的电负性增加时,它们都对Mg~(2+)表现出了更大的亲合力。同时,配体对于Ca~(2+)和Mg~(2+)的区分能力也相应提高。我们的研究结果表明,配体对于Ca~(2+)和Mg~(2+)的区分能力如下:“吲哚-乙酸”协同耦合＞“吲哚-甲酰胺”协同耦合＞“吲哚-吲哚”协同耦合＞“吲哚”耦合。此外,配体的不同结合模式也会影响离子选择性。在“吲哚”耦合和“吲哚-吲哚”协同耦合模式中,吲哚分子结合在第二层时所对应的反应具有最大的离子选择性;而对于“吲哚-乙酸”和“吲哚-甲酰胺”协同耦合模式,吲哚分子结合在第二层,同时单齿脂肪族配体键合第一层时所对应的反应具有最大的离子选择性。更重要的是,我们的计算可用来解释肌质网中Ca~(2+)的选择性机理:当水合Ca~(2+)和水合Mg~(2+)靠近Ca~(2+)-ATP酶时,酶-水界面上的蛋白空穴会优先结合Mg~(2+),导致Mg~(2+)的浓度在局部区域内降低,而这时游离的Ca~(2+)就相对较多。即使有键合的Ca~(2+),其与配体之间较弱的结合力也会使得它们很容易被释放,因此Ca~(2+)可以相对容易地进入Ca~(2+)-ATP酶中的离子传输通道。对于极少数随Ca~(2+)进入通道的Mg~(2+),配体对于Mg~(2+)的强亲合阻止了这个离子的自由传导,而结合较弱的Ca~(2+)则可以以较高的速率通过Ca~(2+)-ATP酶的跨膜区域。此现象与以前报导的“sticky-pore”假说相一致。另外一个影响Ca~(2+)和Mg~(2+)选择性的因素是结合位点空穴的大小和结合位点处的配体在结合阳离子时所能提供的配位数。研究发现,在高亲和位点Ⅰ和Ⅱ处,位点空穴大小和结合位点处的七配位特征更有利于Ca~(2+)的耦合。对于价态不同的Na~+、K~+和Ca~(2+),有如下的几个离子选择性机理(以Na~+和Ca~(2+)为例):第一,一个Ca~(2+)和两个Na~+拥有相同的电荷中和能力,却占有更小的空间,同时由于通道狭小的空间,更倾向于结合Ca~(2+);第二,在传导过程中,若有Na~+、K~+占据配位点,由于Ca~(2+)与配体有更强的相互作用,它可以很容易地取代一价离子,从而占据配位点。第三,配位点处的配体耦合数不适于Na~+的结合。 非键结合模式在钙离子传导过程中的作用非键作用在生命体中广泛存在,而其中的阳离子-π耦合模式在Ca~(2+)-ATP酶、K~+通道和季铵盐通道抑制剂等方面都有重要的作用。因此,我们用二阶微扰的方法计算了与肌质网钙泵和钙通道相关的Ca~(2+)-π相互作用和脂肪族氨基酸参与的Ca~(2+)-π相互作用。计算结果表明,芳香性分子所具有的离域π键特征使得它们成为结合Ca~(2+)的良好配体。而脂肪族氨基酸边链参与反应之后,可以极大地提高Ca~(2+)-配体之间的作用强度。具体来说,对于Ca~(2+)-π复合物,我们研究了Ca~(2+)分别与一到三个苯分子的耦合特征。当Ca~(2+)与两个苯环相互作用时,所对应的最低能量构型是一个交错平行的三明治结构;而当Ca~(2+)与三个苯环作用时,所对应的最低能量构型是一个笼状的结构。很明显,这些结构都具有最小的配体排斥力,并且能够有效地稳定复合物体系。同样的现象也发生在甲酰胺和乙酸耦合的Ca~(2+)-π复合物中。值得注意的是,当甲酰胺直接与苯分子结合时,它对苯环的芳香性特征几乎没有什么影响,而乙酸与苯环的结合则会削弱苯环的芳香性特征。这一点可以从NBO分析和键合轨道分析中反映出来。本章计算结果所体现的最重要的一点是:与脂肪族边链相比,芳香族边链对Ca~(2+)的结合能力更小。而在与肌质网相关的Ca~(2+)传导过程中,达到结合-释放平衡是非常重要的。芳香性分子对Ca~(2+)更弱的亲合使得Ca~(2+)可以更容易从结合的配体中释放出来,从而达到结合-释放的微妙平衡。因此,我们认为芳香性氨基酸在Ca~(2+)传导过程中应该起到了重要的作用。除此之外,Ca~(2+)的结合可以减小蛋白配体的HUMO-LUMO能差,从而导致荧光现象的发生。这一点可以为实验学家们研究Ca~(2+)的结合位点和传导过程提供理论信息和帮助。
[Abstract]:The calcium of skeletal muscle in the endoplasmic reticulum membrane transport is a form of transmission through calcium pump and calcium channels in the process, it has a very important role in the control of muscle contraction and heat, its activity is an important indicator of the size of the various cell energy metabolism and function without damage. Human disorders calcium pump can lead to a variety of diseases, but now the understanding of mechanism of calcium pump and calcium channel is not clear. In addition, the transmembrane calcium ion transfer process remains to be further studied. This paper changes associated with the sarcoplasmic reticulum calcium pump and calcium channel function region molecular conformation the process of calcium ions across the membrane in the organism in the transmission of energy transfer, issues related to interaction of calcium ion and the active site and the ion selectivity, by means of theoretical chemistry in-depth study of the calcium pump and calcium The function principle and the nature of the channel. At the same time, combined with the structure-activity analysis and molecular interaction between all kinds of strength, discusses the effect of amino acid residues, solvent molecules and other environmental factors and structure factors on calcium ion transport mechanism and regulation, and have made some meaningful research results, provide important academic information for the understanding of ion pump and ion channels. The main research results and innovation are summarized as follows:
Ca~ coupling characteristics and biological significance of acidic amino acid and calcium (2+) -ATP enzyme in acidic amino acids in Ca~ (2+) plays an important role in the process of membrane transport, they can be used as ligand coupling directly involved in the reaction, but also can be used to detect Ca~ (2+) with no "detector". Therefore, in this paper, we first examined the system with side chain acid glutamic acid and Ca~ (2+) interaction and its biological significance. Density functional theory study results indicate that in the gas phase, there are 16 kinds of stable -Ca~ glutamic acid (2+) isomers, corresponding glutamate ligands and Ca~ (2+ three) tooth configuration features of bidentate and monodentate coupling. One of the most stable conformation corresponds to Ca~ (2+) and three oxygen atoms (respectively two carboxyl side chain and a backbone carbonyl oxygen) interaction model, and in combination with Ca~ (2+) at the same time a A proton transfer phenomenon. A stable conformation corresponds to a three tooth coupling form (Ca~ (2+) with two carbonyl oxygen and one amino nitrogen interaction), it is only energy structure and energy characteristics of the most stable complex 2.1 kcal/mol. of all complexes from coupling the form is relatively, electrostatic interaction, deformation energy, charge distribution analysis, bonding characters and intramolecular hydrogen bonds were studied. The results showed that glutamic acid three active functional groups with Ca~ (2+) are as follows: the binding ability of the carbonyl oxygen, amino nitrogen, hydroxyl oxygen. In addition, always in the active group of glutamic acid as much as possible with the Ca~ (2+) are coupled. Due to the similarity of structure and energy of the mutual transformation between different complexes are prone to matter. Ca~ real life in the process of muscle (2+) transmembrane conduction details can use our results when the explanation is as follows: Ca~ (2+) -ATPase in E1 configuration, namely Ca~ (2+) with high affinity sites, glutamic acid and Ca~ (2+) coupled with greater intensity, then the corresponding form of the compound for three teeth or double tooth coupling; when Ca~ (2+) transformed into -ATP enzyme with low affinity sites E2 the type -Ca~ (2+), glutamate in unstable form (such as single tooth coupling mode), because the coupling form for Ca~ (2+) from the combination of release in the cavity. In addition, the frequency analysis showed that in the composite, infrared C=O stretching vibration mode with maximum intensity of the absorption peak and, in combination with Ca~ (2+), the peak will shift.
The stepwise hydration effect of glutamate - Ca2 + complexes and their biological significance in actual life, the reaction is carried out in solution or protein environment. And in the solvent molecules, the mode of action of metal ion ligand varies with gas. In order to better close to the real life. We use density functional theory (DFT) of the stepwise hydration on glutamate (2+) -Ca~ system. The influence of thermodynamic parameters of content including hydration, hydration energy and gradually accurate configuration information. At the same time, in order to further investigate the effect of hydration, the charge transfer, bonding and analysis infrared spectra were studied. The results show that the electrostatic interaction, charge transfer, electron orbital effect and ion ligand, influence of ligand ligand repulsion, cut the increase of water molecules Weak Ca-O bond strength, and lead (C-) O-Ca-O (-C) reduce the bond angle. When combined with the water molecules in the Ca~ (2+) of the first layer and the second layer coupling coupling, the periphery of the inner layer of the hydration water molecules near Ca~ (2+), and the inner layer of the carboxyl oxygen gradually away from Ca~ (2+). In addition, due to the increase of hydrogen bonding, the stepwise hydration process can enable glutamate -Ca~ (2+) system becomes more stable. All of the reactions are stepwise hydration enthalpy driven process, but when water molecules are coupled in the first layer, the stepwise hydration process does not continue indefinitely, but a the largest number of water. In this paper the research data show that this number is 6 or 7. in the protein database (PDB), Ca~ (2+) the average coupling number is 6, and the soluble calcium protein in organisms, the most common coupling number is 7, consistent with our results. At the same time. Solvation model The calculation (SCRF) and high level MP2 calculations also confirmed the accuracy of the calculation results. In the calculation process, also found a very important ligand exchange phenomenon, namely: whether the water molecules in the Ca~ coupling (2+) of the first or second layers, with the increase of coupling number, they will the gradual weakening of glutamic acid and Ca~ (2+) the role of strength, until one of the coupling of water molecules instead of glutamate ligand position changed from inner to glutamic acid coupled model for the outer coupling mode. In the real sarcoplasmic reticulum calcium channel, Ca~ (2+) in egg white matter in the void, surrounded by amino acid base: when it via Ca~ (2+) release channel into the cavity, the water molecules in the cavity will replace some or all of the amino acid residues. The original process and the calculation process and is similar, so we can provide the related research Data is used to deepen our understanding of the role of Ca~ (2+) channel. In addition, we also compare the similarity and difference between the computational models and the calcium channels of sarcoplasmic reticulum in real organisms.
The essence of ion selectivity and its influencing factors in Mg~ (2+) concentration ratio of Ca~ (2+) under the condition of high concentration of 1000 times, sarcoplasmic reticulum Ca~ (2+) -ATPase can specifically choose Ca~ (2+) of transmembrane conductance. Ion selective Ca~ (2+) is one of the important characteristics of -ATPase computing. We carried out density functional results show that either aliphatic or aromatic amino acid composition of the protein cavity are easier to combine with Mg~ (2+) and non Ca~ (2+), which is mainly caused by Mg~ (2+) and electrostatic interaction between ligand stronger. Further studies show that for amino acids Heyang water ion affinity characteristics of metal ions, amino acids influence the electronegativity and binding mode and other factors. Compared with Ca~ (2+), Mg~ (2+) has a smaller radius and larger charge density. Therefore, if the binding to the same ligand, Mg~ (2+) will show than Ca~ (2+) stronger electrostatic interaction. On the other hand, when the electronegativity increases, they are Mg~ (2+) showed a greater affinity for the ligand. At the same time, Ca~ (2+) and Mg~ (2+) the ability to distinguish alsoincreased. Our results show that the ligand for Ca~ (2+) and Mg~ (2+) as follows: "the ability to distinguish between indole acetic acid", "cooperative coupling indole carboxamide" synergistic coupling "- > indole indole" > "indole synergistic coupling" coupling. In addition, the ligand binding modes will also affect the ion selectivity in the indole coupling. And "indole indole" synergistic coupling mode, combined with the corresponding indole molecules in the second layer of the reaction is the largest ion selective; the indole acetic acid and indole - formamide "synergistic coupling model, combined with the indole molecules in the second layer, and the single tooth aliphatic ligand bonded article 涓，

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