蛋白質(zhì)在自組裝膜表面吸附的分子模擬研究
發(fā)布時間：2019-03-13 18:02
【摘要】：蛋白質(zhì)在界面的吸附行為在生物醫(yī)用材料、酶固定化等領(lǐng)域都有著廣泛的應(yīng)用。蛋白質(zhì)與界面之間的相互作用研究可以大體分為兩類:1.蛋白質(zhì)的特異性吸附;2.蛋白質(zhì)的非特異性吸附。本論文利用多尺度分子模擬(并行退火蒙特卡洛(parallel tempering Monte Carlo algorithm,PTMC)、粗�；肿觿恿W(xué)(CGMD)和全原子分子動力學(xué)(AAMD))的手段,以模型化的自組裝膜表面為基底,分別對蛋白質(zhì)在帶電表面的吸附取向以及混合帶電自組裝膜的阻抗機理兩大問題進(jìn)行原子水平的模擬研究。擬通過模擬結(jié)果對實驗上新型蛋白質(zhì)固定化材料以及防污抗菌材料的設(shè)計與開發(fā)提供理論指導(dǎo)。主要內(nèi)容和要點如下:1.原型蛋白G B1結(jié)構(gòu)域和變異蛋白G B1結(jié)構(gòu)域在帶電自組裝膜表面呈現(xiàn)出不同的吸附取向。變異蛋白G B1結(jié)構(gòu)域在表面的取向分布相較于原型蛋白G B1結(jié)構(gòu)域而言更集中。這主要是因為原型蛋白質(zhì)本身帶-4 e凈電荷,因此將蛋白中一端的四個帶負(fù)電殘基中和之后,蛋白質(zhì)不僅呈中性,且偶極矩相較于原型蛋白而言更大,因此吸附取向分布更窄。除此之外,我們還發(fā)現(xiàn),盡管改變蛋白G B1結(jié)構(gòu)域的偶極分布可以控制其在帶電表面更有序的吸附,但是吸附后的取向并不利于進(jìn)一步的抗體結(jié)合。而原型蛋白雖然帶有-4e個凈電荷,其依舊可以在表面上以有序的取向吸附,同時,吸附后的取向可以調(diào)控抗體以FAB-up(即抗原結(jié)合域暴露于溶液中)的取向吸附。2.RNase A在負(fù)電表面以活性中心朝向表面的取向吸附,在正電表面以活性中心朝向溶液的取向吸附。RNase A與負(fù)電表面的相互作用相對更強,因此負(fù)電表面可用于除去溶液中多余的RNase A或用于屏蔽RNase A的活性。正電表面由于可調(diào)控RNase A以活性中心暴露的取向吸附,因此可用于RNase A的固定化。RNase A在帶電表面吸附的過程中,偶極矩發(fā)生微小變化,但是蛋白質(zhì)的整體骨架結(jié)構(gòu)基本保留。也就是說,弱帶電表面并不影響RNase A的整體天然構(gòu)象。3.阿魏酸酯酶在帶電自組裝膜表面的吸附行為受到表面的帶電性質(zhì)、表面電荷密度以及溶液離子強度等因素影響。通過模擬發(fā)現(xiàn)阿魏酸酯酶在帶電表面的吸附行為主要靠酶與表面之間的靜電相互作用能決定。溶液中離子強度增大時,阿魏酸酯酶與帶電表面之間的靜電相互作用會減弱。當(dāng)阿魏酸酯酶吸附在表面電荷密度較小的正電表面且溶液中離子濃度較大時,阿魏酸酯酶的取向最有利于其催化活性。界面的反號離子層在阿魏酸酯酶吸附到負(fù)電表面的過程中起著不可忽視的作用。在本工作涉及到的表面電荷密度和溶液離子強度條件下,阿魏酸酯酶都完好地保留了其天然構(gòu)象。4.漆酶在電極表面的吸附取向直接關(guān)系到酶固定化電極的直接電子傳導(dǎo)效率。對于漆酶而言,T1中心靠近表面更有利于直接電子傳導(dǎo)。本工作的結(jié)論與已有的實驗結(jié)果相一致,都證實正電表面更適合于漆酶的固定化。通過模擬發(fā)現(xiàn),漆酶在正電表面以‘end-on’取向吸附,在負(fù)電表面以‘lying’取向吸附。當(dāng)吸附到正電表面時,漆酶的T1中心相對于負(fù)電表面的漆酶的T1中心更接近表面,同時漆酶在正電表面的吸附相比于負(fù)電表面更穩(wěn)定。5.針對COO-封端烷基硫醇上的羧基存在酯化和水解兩種狀態(tài),通過多尺度分子模擬研究了COO-/N(CH3)3+-封端烷基硫醇混合帶電自組裝膜與纖維蛋白原(gamma fibrinogen,γFg)之間的相互作用機制以及水解反應(yīng)對γFg吸附行為的影響�；旌蠋щ娮越M裝膜中兩種分子鏈的比例是1:1。模擬發(fā)現(xiàn),經(jīng)過水解反應(yīng),COOCH3-封端烷基硫醇(電中性)轉(zhuǎn)化為COO--封端烷基硫醇(帶-1 e電荷)混合帶電自組裝膜發(fā)生了從抗菌性質(zhì)到阻抗性質(zhì)的轉(zhuǎn)變。模擬發(fā)現(xiàn)水解前后,混合帶電自組裝膜表面發(fā)生的最主要的變化是表面的帶電性質(zhì)和界面的水化層。γFg能夠穩(wěn)定地吸附在水解前帶正電的COOCH3-/N(CH3)3+-封端自組裝膜表面,吸附過程主要是由靜電相互作用誘導(dǎo)。γFg與COOCH3-/N(CH3)3+-封端自組裝膜表面上方的單層水化層之間的范德華相互作用能不足以抵抗γFg與COOCH3-/N(CH3)3+-封端自組裝膜表面之間強的靜電相互作用能。水解之后,帶正電的自組裝膜表面轉(zhuǎn)化為中性的混合帶電自組裝膜,γFg與表面之間的靜電相互作用能消失。同時,自組裝膜表面被“雙層水化層”覆蓋,該“雙層水化層”是由N(CH3)3+和COO-基團共同誘導(dǎo)產(chǎn)生的。通過“雙層水化層”和靜電相互作用能消失兩個因素的共同作用,使得γFg從表面解析。除此之外,水解之后,由于N(CH3)3+和COO-基團直接的靜電相互作用,自組裝膜的結(jié)構(gòu)更規(guī)整了。
[Abstract]:The adsorption behavior of the protein in the interface has wide application in the fields of biological medical material, enzyme immobilization and the like. The interaction between the protein and the interface can be divided into two categories:1. The specific adsorption of proteins;2. The non-specific adsorption of the protein. By means of multi-scale molecular simulation (parallel annealing Monte Carlo algorithm, PTMC), coarse-grained molecular dynamics (CGMD) and full-atomic molecular dynamics (AAMD), the surface of the self-assembled membrane is modeled as a substrate. The adsorption orientation of the protein on the charged surface and the impedance mechanism of the mixed charged self-assembled membrane were studied. It is proposed to provide theoretical guidance for the design and development of novel protein-immobilized materials and anti-fouling antibacterial materials in the experiment. The main contents and points are as follows:1. The prototype protein G B1 domain and the variant protein G B1 domain exhibit different adsorption orientations on the surface of the charged self-assembled membrane. The orientation distribution of the variant protein G B1 domain on the surface is more concentrated than the prototype protein G B1 domain. This is mainly due to the net charge of the prototype protein itself-4e, thus neutralizing the four negatively charged residues at one end of the protein, the protein is not only neutral, and the dipole moment is larger than the prototype protein, so the adsorption orientation distribution is narrower. In addition, we have also found that, although the dipole distribution of the altered protein G B1 domain can control its more orderly adsorption on the charged surface, the post-adsorption orientation is not detrimental to further antibody binding. while the prototype protein has a net charge of-4e, it can still be adsorbed on the surface in an ordered orientation, After the adsorption, the orientation of the antibody is adsorbed by the orientation of FAB-up (i.e., the antigen-binding domain is exposed to the solution). The interaction of the RNase A with the negative surface is relatively stronger, so that the negative surface can be used to remove excess RNase A from the solution or to shield the activity of the RNase A. The positively charged surface can be used for the immobilization of the RNase A, since it can regulate the orientation and adsorption of the RNase A in the active center. In the process of adsorption of the charged surface, the dipole moment is slightly changed, but the overall skeleton structure of the protein is basically preserved. That is, the weakly charged surface does not affect the overall natural conformation of the RNase A. The adsorption behavior of the feruloyl esterase on the surface of the charged self-assembled membrane is influenced by the charged property of the surface, the surface charge density and the ionic strength of the solution. It is found that the adsorption behavior of feruloyl esterase on the charged surface mainly depends on the electrostatic interaction between the enzyme and the surface. When the ionic strength in the solution is increased, the electrostatic interaction between the feruloyl esterase and the charged surface is reduced. When the feruloyl esterase is adsorbed on the positive surface with a small surface charge density and the concentration of the ions in the solution is large, the orientation of the feruloyl esterase is most favorable for the catalytic activity. The anti-ionic layer of the interface plays an important role in the adsorption of feruloyl esterase to the negative surface. Ferulic acid esterase kept its natural conformation in good condition under the surface charge density and the ionic strength of the solution. The adsorption orientation of the laccase on the surface of the electrode is directly related to the direct electron-conduction efficiency of the enzyme-immobilized electrode. For laccase, the T1 center is closer to the surface to facilitate direct electron conduction. The conclusion of this work is consistent with the existing experimental results, and it is confirmed that the positive surface is more suitable for the immobilization of the laccase. It was found that the laccase was adsorbed on the positively charged surface with the "end-on 'orientation, and the negative surface was adsorbed by" lysing' orientation. When the positive surface is adsorbed, the T1 center of the laccase is closer to the surface than the T1 center of the laccase of the negative surface, while the adsorption of the laccase on the positive surface is more stable than the negative surface. The co-charged self-assembled membrane and fibrinogen (gamma fibringen) of COO-/ N (CH3)3 +-terminated alkyl mercaptan were studied by multi-scale molecular simulation. The interaction mechanism between (Fg) and the effect of the hydrolysis reaction on the adsorption behavior of Fg. The ratio of the two molecular chains in the mixed charged self-assembled film is 1:1. It has been found that the conversion of COCH3-terminated alkyl mercaptan (electroneutral) to COO--terminated alkyl mercaptan (with-1e charge) has taken place from the antibacterial property to the impedance property after the hydrolysis reaction. The most important change of the surface of the mixed charged self-assembled membrane before and after the hydrolysis is the surface's electrification property and the hydration layer of the interface. The HCFg can stably adsorb the positively charged COCH3-/ N (CH3)3 +-terminated self-assembled membrane surface before hydrolysis, and the adsorption process is mainly induced by electrostatic interaction. The Van der Waals interaction between the Fg and the COCH3-/ N (CH3)3 +-terminated self-assembled film surface can not be sufficient to resist the strong electrostatic interaction between the surface Fg and the COCH3-/ N (CH3)3 +-terminated self-assembled film surface. After the hydrolysis, the positively charged self-assembled membrane surface is converted into a neutral, mixed charged self-assembled film, and the electrostatic interaction between the surface Fg and the surface can be eliminated. At the same time, the self-assembled film surface is covered by a double-layer hydration layer, which is co-induced by N (CH3)3 + and COO-groups. The co-action of the two factors can be eliminated by the interaction of the "double-layer hydration layer" and the electrostatic, so that the envelope Fg is resolved from the surface. In addition, after hydrolysis, the structure of the self-assembled film is more regular due to the direct electrostatic interaction of the N (CH3)3 + and the COO-group.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：O629.73;O647.3
本文編號：2439630
[Abstract]:The adsorption behavior of the protein in the interface has wide application in the fields of biological medical material, enzyme immobilization and the like. The interaction between the protein and the interface can be divided into two categories:1. The specific adsorption of proteins;2. The non-specific adsorption of the protein. By means of multi-scale molecular simulation (parallel annealing Monte Carlo algorithm, PTMC), coarse-grained molecular dynamics (CGMD) and full-atomic molecular dynamics (AAMD), the surface of the self-assembled membrane is modeled as a substrate. The adsorption orientation of the protein on the charged surface and the impedance mechanism of the mixed charged self-assembled membrane were studied. It is proposed to provide theoretical guidance for the design and development of novel protein-immobilized materials and anti-fouling antibacterial materials in the experiment. The main contents and points are as follows:1. The prototype protein G B1 domain and the variant protein G B1 domain exhibit different adsorption orientations on the surface of the charged self-assembled membrane. The orientation distribution of the variant protein G B1 domain on the surface is more concentrated than the prototype protein G B1 domain. This is mainly due to the net charge of the prototype protein itself-4e, thus neutralizing the four negatively charged residues at one end of the protein, the protein is not only neutral, and the dipole moment is larger than the prototype protein, so the adsorption orientation distribution is narrower. In addition, we have also found that, although the dipole distribution of the altered protein G B1 domain can control its more orderly adsorption on the charged surface, the post-adsorption orientation is not detrimental to further antibody binding. while the prototype protein has a net charge of-4e, it can still be adsorbed on the surface in an ordered orientation, After the adsorption, the orientation of the antibody is adsorbed by the orientation of FAB-up (i.e., the antigen-binding domain is exposed to the solution). The interaction of the RNase A with the negative surface is relatively stronger, so that the negative surface can be used to remove excess RNase A from the solution or to shield the activity of the RNase A. The positively charged surface can be used for the immobilization of the RNase A, since it can regulate the orientation and adsorption of the RNase A in the active center. In the process of adsorption of the charged surface, the dipole moment is slightly changed, but the overall skeleton structure of the protein is basically preserved. That is, the weakly charged surface does not affect the overall natural conformation of the RNase A. The adsorption behavior of the feruloyl esterase on the surface of the charged self-assembled membrane is influenced by the charged property of the surface, the surface charge density and the ionic strength of the solution. It is found that the adsorption behavior of feruloyl esterase on the charged surface mainly depends on the electrostatic interaction between the enzyme and the surface. When the ionic strength in the solution is increased, the electrostatic interaction between the feruloyl esterase and the charged surface is reduced. When the feruloyl esterase is adsorbed on the positive surface with a small surface charge density and the concentration of the ions in the solution is large, the orientation of the feruloyl esterase is most favorable for the catalytic activity. The anti-ionic layer of the interface plays an important role in the adsorption of feruloyl esterase to the negative surface. Ferulic acid esterase kept its natural conformation in good condition under the surface charge density and the ionic strength of the solution. The adsorption orientation of the laccase on the surface of the electrode is directly related to the direct electron-conduction efficiency of the enzyme-immobilized electrode. For laccase, the T1 center is closer to the surface to facilitate direct electron conduction. The conclusion of this work is consistent with the existing experimental results, and it is confirmed that the positive surface is more suitable for the immobilization of the laccase. It was found that the laccase was adsorbed on the positively charged surface with the "end-on 'orientation, and the negative surface was adsorbed by" lysing' orientation. When the positive surface is adsorbed, the T1 center of the laccase is closer to the surface than the T1 center of the laccase of the negative surface, while the adsorption of the laccase on the positive surface is more stable than the negative surface. The co-charged self-assembled membrane and fibrinogen (gamma fibringen) of COO-/ N (CH3)3 +-terminated alkyl mercaptan were studied by multi-scale molecular simulation. The interaction mechanism between (Fg) and the effect of the hydrolysis reaction on the adsorption behavior of Fg. The ratio of the two molecular chains in the mixed charged self-assembled film is 1:1. It has been found that the conversion of COCH3-terminated alkyl mercaptan (electroneutral) to COO--terminated alkyl mercaptan (with-1e charge) has taken place from the antibacterial property to the impedance property after the hydrolysis reaction. The most important change of the surface of the mixed charged self-assembled membrane before and after the hydrolysis is the surface's electrification property and the hydration layer of the interface. The HCFg can stably adsorb the positively charged COCH3-/ N (CH3)3 +-terminated self-assembled membrane surface before hydrolysis, and the adsorption process is mainly induced by electrostatic interaction. The Van der Waals interaction between the Fg and the COCH3-/ N (CH3)3 +-terminated self-assembled film surface can not be sufficient to resist the strong electrostatic interaction between the surface Fg and the COCH3-/ N (CH3)3 +-terminated self-assembled film surface. After the hydrolysis, the positively charged self-assembled membrane surface is converted into a neutral, mixed charged self-assembled film, and the electrostatic interaction between the surface Fg and the surface can be eliminated. At the same time, the self-assembled film surface is covered by a double-layer hydration layer, which is co-induced by N (CH3)3 + and COO-groups. The co-action of the two factors can be eliminated by the interaction of the "double-layer hydration layer" and the electrostatic, so that the envelope Fg is resolved from the surface. In addition, after hydrolysis, the structure of the self-assembled film is more regular due to the direct electrostatic interaction of the N (CH3)3 + and the COO-group.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：O629.73;O647.3
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 章愛娟;謝韻;周健;;蛋白質(zhì)界面取向的實驗控制與表征[J];化學(xué)進(jìn)展;2009年Z2期
2 蘭惠清,張嗣偉,王德國;探針作用下自組裝膜的分子動力學(xué)模擬[J];自然科學(xué)進(jìn)展;2002年06期
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