蛋白质在自组装膜表面吸附的分子模拟研究
发布时间：2019-03-13 18:02
【摘要】：蛋白质在界面的吸附行为在生物医用材料、酶固定化等领域都有着广泛的应用。蛋白质与界面之间的相互作用研究可以大体分为两类:1.蛋白质的特异性吸附;2.蛋白质的非特异性吸附。本论文利用多尺度分子模拟(并行退火蒙特卡洛(parallel tempering Monte Carlo algorithm,PTMC)、粗粒化分子动力学(CGMD)和全原子分子动力学(AAMD))的手段,以模型化的自组装膜表面为基底,分别对蛋白质在带电表面的吸附取向以及混合带电自组装膜的阻抗机理两大问题进行原子水平的模拟研究。拟通过模拟结果对实验上新型蛋白质固定化材料以及防污抗菌材料的设计与开发提供理论指导。主要内容和要点如下:1.原型蛋白G B1结构域和变异蛋白G B1结构域在带电自组装膜表面呈现出不同的吸附取向。变异蛋白G B1结构域在表面的取向分布相较于原型蛋白G B1结构域而言更集中。这主要是因为原型蛋白质本身带-4 e净电荷,因此将蛋白中一端的四个带负电残基中和之后,蛋白质不仅呈中性,且偶极矩相较于原型蛋白而言更大,因此吸附取向分布更窄。除此之外,我们还发现,尽管改变蛋白G B1结构域的偶极分布可以控制其在带电表面更有序的吸附,但是吸附后的取向并不利于进一步的抗体结合。而原型蛋白虽然带有-4e个净电荷,其依旧可以在表面上以有序的取向吸附,同时,吸附后的取向可以调控抗体以FAB-up(即抗原结合域暴露于溶液中)的取向吸附。2.RNase A在负电表面以活性中心朝向表面的取向吸附,在正电表面以活性中心朝向溶液的取向吸附。RNase A与负电表面的相互作用相对更强,因此负电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。正电表面由于可调控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在带电表面吸附的过程中,偶极矩发生微小变化,但是蛋白质的整体骨架结构基本保留。也就是说,弱带电表面并不影响RNase A的整体天然构象。3.阿魏酸酯酶在带电自组装膜表面的吸附行为受到表面的带电性质、表面电荷密度以及溶液离子强度等因素影响。通过模拟发现阿魏酸酯酶在带电表面的吸附行为主要靠酶与表面之间的静电相互作用能决定。溶液中离子强度增大时,阿魏酸酯酶与带电表面之间的静电相互作用会减弱。当阿魏酸酯酶吸附在表面电荷密度较小的正电表面且溶液中离子浓度较大时,阿魏酸酯酶的取向最有利于其催化活性。界面的反号离子层在阿魏酸酯酶吸附到负电表面的过程中起着不可忽视的作用。在本工作涉及到的表面电荷密度和溶液离子强度条件下,阿魏酸酯酶都完好地保留了其天然构象。4.漆酶在电极表面的吸附取向直接关系到酶固定化电极的直接电子传导效率。对于漆酶而言,T1中心靠近表面更有利于直接电子传导。本工作的结论与已有的实验结果相一致,都证实正电表面更适合于漆酶的固定化。通过模拟发现,漆酶在正电表面以‘end-on’取向吸附,在负电表面以‘lying’取向吸附。当吸附到正电表面时,漆酶的T1中心相对于负电表面的漆酶的T1中心更接近表面,同时漆酶在正电表面的吸附相比于负电表面更稳定。5.针对COO-封端烷基硫醇上的羧基存在酯化和水解两种状态,通过多尺度分子模拟研究了COO-/N(CH3)3+-封端烷基硫醇混合带电自组装膜与纤维蛋白原(gamma fibrinogen,γFg)之间的相互作用机制以及水解反应对γFg吸附行为的影响。混合带电自组装膜中两种分子链的比例是1:1。模拟发现,经过水解反应,COOCH3-封端烷基硫醇(电中性)转化为COO--封端烷基硫醇(带-1 e电荷)混合带电自组装膜发生了从抗菌性质到阻抗性质的转变。模拟发现水解前后,混合带电自组装膜表面发生的最主要的变化是表面的带电性质和界面的水化层。γFg能够稳定地吸附在水解前带正电的COOCH3-/N(CH3)3+-封端自组装膜表面,吸附过程主要是由静电相互作用诱导。γFg与COOCH3-/N(CH3)3+-封端自组装膜表面上方的单层水化层之间的范德华相互作用能不足以抵抗γFg与COOCH3-/N(CH3)3+-封端自组装膜表面之间强的静电相互作用能。水解之后,带正电的自组装膜表面转化为中性的混合带电自组装膜,γFg与表面之间的静电相互作用能消失。同时,自组装膜表面被“双层水化层”覆盖,该“双层水化层”是由N(CH3)3+和COO-基团共同诱导产生的。通过“双层水化层”和静电相互作用能消失两个因素的共同作用,使得γFg从表面解析。除此之外,水解之后,由于N(CH3)3+和COO-基团直接的静电相互作用,自组装膜的结构更规整了。
[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.
【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：O629.73;O647.3
本文编号：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.
【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：O629.73;O647.3
【参考文献】
相关期刊论文 前2条
1 章爱娟;谢韵;周健;;蛋白质界面取向的实验控制与表征[J];化学进展;2009年Z2期
2 兰惠清,张嗣伟,王德国;探针作用下自组装膜的分子动力学模拟[J];自然科学进展;2002年06期
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