發(fā)布時間：2018-01-07 19:18

  本文關鍵詞：肌質網鈣泵作用機理的相關問題研究　出處：《山東大學》2008年博士論文　論文類型：學位論文

  更多相關文章： 肌質網鈣泵 鈣通道 耦合作用模式 環(huán)境因素影響 離子選擇性 鈣離子跨膜傳輸機理

【摘要】： 骨骼肌內質網中的鈣離子跨膜傳輸是一個通過鈣泵和鈣離子通道的形式傳輸鈣離子的過程,它在控制肌肉收縮和生熱方面都有非常重要的作用,其活性的大小是各種細胞能量代謝及功能有無損傷的重要指標。人體內鈣泵的失調可以導致多種疾病的發(fā)生,但現(xiàn)在對于鈣泵和鈣通道的作用機制尚無明確的認識。此外,鈣離子的跨膜傳輸過程還有待于進一步的考察。本論文圍繞著生物體中鈣離子跨膜傳輸過程中所涉及的與肌質網鈣泵和鈣通道有關的功能區(qū)域分子構象的變化、能量傳遞、鈣離子與各活性位點的耦合作用以及離子選擇性等有關問題,用理論化學方法深入研究了鈣泵和鈣通道的功能原理和本質。同時,結合構效分析及分子間各類強弱相互作用的研究,探討了氨基酸殘基、溶劑分子等環(huán)境因素和結構因素等對鈣離子傳輸機制的影響和調控作用,并取得了一些有意義的研究成果,為認識離子泵和離子通道提供了重要的理論信息。主要的研究成果與創(chuàng)新簡述如下: 酸性氨基酸與鈣離子的耦合特征及其生物意義Ca~(2+)-ATP酶中的酸性氨基酸在Ca~(2+)跨膜傳輸過程中有重要的作用,它們既可以作為耦合配體直接參與反應,又可作為探測Ca~(2+)結合與否的“探測器”。因此,在本論文中,我們首先系統(tǒng)考察了具有酸性邊鏈的谷氨酸分子和Ca~(2+)的相互作用及其生物意義。密度泛函理論的研究結果表明,在氣相中,存在16種穩(wěn)定的谷氨酸-Ca~(2+)異構體,分別對應著谷氨酸配體和Ca~(2+)的三齒、雙齒和單齒耦合的構型特征。其中最穩(wěn)定的構象對應著Ca~(2+)與三個氧原子(分別為邊鏈的兩個羧基氧和一個主鏈羰基氧)相互作用的模式,并且在結合Ca~(2+)的同時伴隨著一個質子轉移的現(xiàn)象。次穩(wěn)定的構象也對應著一個三齒耦合的形式(Ca~(2+)與兩個羰基氧和一個氨基氮相互作用),它的能量僅比最穩(wěn)定的復合物高2.1 kcal/mol。所有復合物的構型和能量特征都從耦合形式、相對能、靜電作用、形變能、電荷布局分析、軌道鍵合特征和分子內氫鍵等方面進行了研究。結果表明,谷氨酸的三個活性官能團與Ca~(2+)的結合能力如下:羰基氧＞氨基氮＞羥基氧。另外,谷氨酸總是以盡可能多的活性基團與Ca~(2+)進行耦合。由于構型和能量上的相似性,不同復合物之間的相互轉化也極易發(fā)生。實際生命過程中的肌質網Ca~(2+)跨膜傳導細節(jié)可以用我們的計算結果解釋如下:當Ca~(2+)-ATPase處于E1構型,即Ca~(2+)結合在高親和位點時,谷氨酸會與Ca~(2+)以較大的強度進行耦合,此時對應的復合物形式為三齒或雙齒耦合;當Ca~(2+)-ATP酶轉化到具有低親和位點的E2構型時,谷氨酸-Ca~(2+)會以不穩(wěn)定的形式(如單齒耦合模式)存在,因為此種耦合形式有利于Ca~(2+)從結合位釋放到內腔中。此外,頻率分析表明,在復合物中,C=O伸縮振動模式具有最大強度的紅外吸收峰,并且在結合了Ca~(2+)之后,這些峰都會紅移。 谷氨酸-鈣離子復合物的逐步水合效應及其生物意義在實際的生命體中,反應都是在溶液或蛋白環(huán)境下進行的。而在溶劑分子的作用下,金屬離子-配體的作用模式會與氣態(tài)中有所區(qū)別。為了更好地接近于真實的生命過程,我們又采用密度泛函理論(DFT)研究了逐步水合對谷氨酸-Ca~(2+)體系的影響。研究內容包括水合反應的熱力學參數(shù),逐步水合能及精確的構型信息等。同時,為了進一步考察水合作用的影響,本文也對相關的電荷轉移、鍵合分析以及紅外光譜特征進行了研究。計算結果表明,在靜電作用、電荷轉移、電子軌道效應和離子-配體、配體-配體排斥力的共同影響下,水分子的增加削弱了Ca-O鍵的強度,并導致了(C-)O-Ca-O(-C)鍵角的減小。而當水分子同時結合在Ca~(2+)的第一耦合層和第二耦合層時,外圍的水合使得內層水分子靠近Ca~(2+),而內層的羧基氧逐漸遠離Ca~(2+)。此外,由于氫鍵作用的增加,逐步水合過程可以使谷氨酸-Ca~(2+)體系變得更穩(wěn)定。所有的逐步水合反應均為焓驅動過程,但是當耦合的水分子位于第一層時,這種逐步水合過程并不能無限制地進行下去,而是有一個最大的水合數(shù)。本文的研究數(shù)據(jù)表明此數(shù)值為6或7。在蛋白質數(shù)據(jù)庫(PDB)中,Ca~(2+)的平均耦合數(shù)也為6,而在可溶性的生物體鈣蛋白中,最常見的耦合數(shù)為7,與我們的計算結果相一致。同時,溶劑化模型計算(SCRF)和高水平的MP2計算也證實了計算結果的準確性。在計算過程中,還發(fā)現(xiàn)了一個非常重要的配體交換的現(xiàn)象,即:無論水分子耦合在Ca~(2+)的第一層還是第二層,隨著耦合數(shù)的增加,它們都會逐步削弱谷氨酸和Ca~(2+)的作用強度,直至其中的一個耦合水分子代替谷氨酸配體的位置,使谷氨酸由內層耦合模式轉變?yōu)橥鈱玉詈夏Ｊ�。在真實的肌質網鈣通道中,Ca~(2+)處于蛋白質空穴中,為氨基酸殘基所包圍:而當其經由Ca~(2+)釋放通道進入內腔時,內腔中的水分子會部分或全部代替原來的氨基酸殘基。此過程與我們的模擬計算過程是及其類似的,因此我們的研究完全可以提供相關的理論數(shù)據(jù)來加深人們對于Ca~(2+)通道作用的理解。另外,我們還對所采用的計算模型與實際生物體中肌質網鈣通道的相似性和不同點進行了比較研究。 離子選擇性的本質及其影響因素在Mg~(2+)濃度比Ca~(2+)濃度高數(shù)千倍的情況下,肌質網Ca~(2+)-ATPase仍可以特異性地選擇Ca~(2+)進行跨膜傳導。離子選擇性是Ca~(2+)-ATPase的一個重要特征。我們所進行的密度泛函的計算結果表明,無論是由脂肪族還是芳香族氨基酸組成的蛋白空穴都更容易結合Mg~(2+)而非Ca~(2+),這主要是由Mg~(2+)和配體之間更強的靜電相互作用引起的。進一步的研究表明,氨基酸對于水合陽離子的親合力受金屬離子的特性、氨基酸配體的電負性和結合模式等因素的影響。相對于Ca~(2+)來說,Mg~(2+)有更小的半徑和更大的電荷密度。因此,如果結合到同一個配體上,Mg~(2+)會表現(xiàn)出比Ca~(2+)更強的靜電相互作用。另一方面,當配體的電負性增加時,它們都對Mg~(2+)表現(xiàn)出了更大的親合力。同時,配體對于Ca~(2+)和Mg~(2+)的區(qū)分能力也相應提高。我們的研究結果表明,配體對于Ca~(2+)和Mg~(2+)的區(qū)分能力如下:“吲哚-乙酸”協(xié)同耦合＞“吲哚-甲酰胺”協(xié)同耦合＞“吲哚-吲哚”協(xié)同耦合＞“吲哚”耦合。此外,配體的不同結合模式也會影響離子選擇性。在“吲哚”耦合和“吲哚-吲哚”協(xié)同耦合模式中,吲哚分子結合在第二層時所對應的反應具有最大的離子選擇性;而對于“吲哚-乙酸”和“吲哚-甲酰胺”協(xié)同耦合模式,吲哚分子結合在第二層,同時單齒脂肪族配體鍵合第一層時所對應的反應具有最大的離子選擇性。更重要的是,我們的計算可用來解釋肌質網中Ca~(2+)的選擇性機理:當水合Ca~(2+)和水合Mg~(2+)靠近Ca~(2+)-ATP酶時,酶-水界面上的蛋白空穴會優(yōu)先結合Mg~(2+),導致Mg~(2+)的濃度在局部區(qū)域內降低,而這時游離的Ca~(2+)就相對較多。即使有鍵合的Ca~(2+),其與配體之間較弱的結合力也會使得它們很容易被釋放,因此Ca~(2+)可以相對容易地進入Ca~(2+)-ATP酶中的離子傳輸通道。對于極少數(shù)隨Ca~(2+)進入通道的Mg~(2+),配體對于Mg~(2+)的強親合阻止了這個離子的自由傳導,而結合較弱的Ca~(2+)則可以以較高的速率通過Ca~(2+)-ATP酶的跨膜區(qū)域。此現(xiàn)象與以前報導的“sticky-pore”假說相一致。另外一個影響Ca~(2+)和Mg~(2+)選擇性的因素是結合位點空穴的大小和結合位點處的配體在結合陽離子時所能提供的配位數(shù)。研究發(fā)現(xiàn),在高親和位點Ⅰ和Ⅱ處,位點空穴大小和結合位點處的七配位特征更有利于Ca~(2+)的耦合。對于價態(tài)不同的Na~+、K~+和Ca~(2+),有如下的幾個離子選擇性機理(以Na~+和Ca~(2+)為例):第一,一個Ca~(2+)和兩個Na~+擁有相同的電荷中和能力,卻占有更小的空間,同時由于通道狹小的空間,更傾向于結合Ca~(2+);第二,在傳導過程中,若有Na~+、K~+占據(jù)配位點,由于Ca~(2+)與配體有更強的相互作用,它可以很容易地取代一價離子,從而占據(jù)配位點。第三,配位點處的配體耦合數(shù)不適于Na~+的結合。 非鍵結合模式在鈣離子傳導過程中的作用非鍵作用在生命體中廣泛存在,而其中的陽離子-π耦合模式在Ca~(2+)-ATP酶、K~+通道和季銨鹽通道抑制劑等方面都有重要的作用。因此,我們用二階微擾的方法計算了與肌質網鈣泵和鈣通道相關的Ca~(2+)-π相互作用和脂肪族氨基酸參與的Ca~(2+)-π相互作用。計算結果表明,芳香性分子所具有的離域π鍵特征使得它們成為結合Ca~(2+)的良好配體。而脂肪族氨基酸邊鏈參與反應之后,可以極大地提高Ca~(2+)-配體之間的作用強度。具體來說,對于Ca~(2+)-π復合物,我們研究了Ca~(2+)分別與一到三個苯分子的耦合特征。當Ca~(2+)與兩個苯環(huán)相互作用時,所對應的最低能量構型是一個交錯平行的三明治結構;而當Ca~(2+)與三個苯環(huán)作用時,所對應的最低能量構型是一個籠狀的結構。很明顯,這些結構都具有最小的配體排斥力,并且能夠有效地穩(wěn)定復合物體系。同樣的現(xiàn)象也發(fā)生在甲酰胺和乙酸耦合的Ca~(2+)-π復合物中。值得注意的是,當甲酰胺直接與苯分子結合時,它對苯環(huán)的芳香性特征幾乎沒有什么影響,而乙酸與苯環(huán)的結合則會削弱苯環(huán)的芳香性特征。這一點可以從NBO分析和鍵合軌道分析中反映出來。本章計算結果所體現(xiàn)的最重要的一點是:與脂肪族邊鏈相比,芳香族邊鏈對Ca~(2+)的結合能力更小。而在與肌質網相關的Ca~(2+)傳導過程中,達到結合-釋放平衡是非常重要的。芳香性分子對Ca~(2+)更弱的親合使得Ca~(2+)可以更容易從結合的配體中釋放出來,從而達到結合-釋放的微妙平衡。因此,我們認為芳香性氨基酸在Ca~(2+)傳導過程中應該起到了重要的作用。除此之外,Ca~(2+)的結合可以減小蛋白配體的HUMO-LUMO能差,從而導致熒光現(xiàn)象的發(fā)生。這一點可以為實驗學家們研究Ca~(2+)的結合位點和傳導過程提供理論信息和幫助。
[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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