【作者】 吕正检；

【导师】 陈国平； 王建华；

【作者基本信息】 重庆大学 ， 生物医学工程， 2010， 博士

【摘要】 蛋白质是生命的基础,它是生物体内一切功能活动的主要执行者。生物体内的机制大部分是经由蛋白质与蛋白质之间的相互作用而发挥生理功能。基于抗原或抗体的蛋白质分子间的相互作用研究是蛋白质分子间相作用研究的重要领域。本文以胰岛素(INS)和胰岛素降解酶(IDE)、人免疫球蛋白G(人IgG,以下简称抗原)和大鼠抗人IgG蛋白质(以下简称抗体)为模型系统,主要进行了以下几个方面的研究:(1)采用用于生物系统相互作用的二维图形学实验室(2D-GraLab)的分子模拟方法对INS(PDB序列号2jv1)和IDE(PDB序列号2jg4)相互作用进行模拟,以谋求获得蛋白质间相互作用的形式和内涵。结果显示INS和IDE的结合过程中主要存在着溶剂化效应和范德华力相互作用。其中,复合物A链和B链对结合的溶剂化自由能分别做出了-4.288 kcal/mol和-5.495 kcal/mol的贡献。而复合物A链和B链对结合的范德华力相互作用分别做出了-0.199 kcal/mol和-0.249 kcal/mol的贡献。同时,由残基配对总结图中可知,INS的A链55位Thr残基和B链的30位Thr残基之间发生了立体碰撞,其质心间距离为1.71 nm。INS的A链53位His残基和B链4位的Glu残基发生了离子对相互作用,其质心间距离为5.35 nm。INS界面由疏水性的氨基酸组成,由此形成的强疏水性导致了其在溶液环境中的不稳定性,从而作为一种有效的化学力推动了该分子与IDE发生纳摩尔水平上的结合。IDE和INS在结合界面上发生了分子表面间的密集接触,造成了一个明显的基质镶嵌位点,并形成了一对静电作用的盐桥。与此同时,在范德华形状互补和疏水驱动的协同作用力下,IDE和INS之间瞬时形成了稳定的复合物结构,从而介导下游生物学效应。(2)运用自组装(SAM)方法制备了16-巯基棕榈酸(MHA)分子膜,并将抗体分子共价连接在经1-乙基-3-(3-二甲基氨基丙基)-碳化二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)活化后的MHA膜上,从而实现了抗体的固定,获得了均一可控的抗体单分子层。对制得的抗体单分子层分别采用轻敲模式原子力显微镜(TM-AFM)、掠入射X射线衍射(GIXRD)、X射线光电子能谱(XPS)、接触角(CA)测试等方法对其表征。空白金片、MHA膜和抗体单分子层的二维和三维形貌显示三种不同表面具有截然不同的微结构。GIXRD测试显示MHA膜和抗体单分子层的GIXRD图谱与空白金片显著不同,均在0-150的2θ角范围内有特征峰,但MHA膜和抗体单分子层的GIXRD的峰形及峰位并不一致且具有明显差异。空白金片、MHA膜和抗体单分子层的XPS测试结果表明三种表面的元素组成均与预期的吻合,空白金片的Au结合能图谱与标准图谱一致,在86.6 eV和82.9 eV处存在强峰,经MHA修饰后的金衬底其Au结合能图谱峰位发生了化学位移(移至87.46 eV和91.11 eV处)。MHA膜的S2p结合能图谱在162.17 eV和161 eV处有强峰。在S2p结合能图谱中未发现高于164 eV的峰,意味着制得的MHA膜上不存在游离的MHA分子。抗体单分子层的的N1s结合能图谱在400.55 eV有强峰,提示抗体分子已成功地连接在MHA膜上。CA测试结果显示MHA膜和抗体单分子层的接触角分别为180和140,均具有十分亲水的表面。此外,本文还测试了MHA与正十二硫醇一系列摩尔比的混合硫醇分子膜的接触角,结果发现接触角随着MHA比例的增大而减小,呈反相关关系,而随着正十二硫醇的所占的比例增大而增大,呈正相关关系。(3)采用TM-AFM和摩擦力显微镜(FFM)对抗原和抗体分子间的相互作用进行成像学研究。在两种成像方法中,分别对空白金片、抗体单分子层以及抗原/抗体复合物分子层进行成像。在TM-AFM研究中,同时记录其表面形貌图和相位图,对比形貌图和相位图可见三种不同表面的具有不同的微结构,形貌图与相位图互为呼应与验证,表明抗体分子已成功固定在硫醇修饰的金衬底上,抗体与抗原之间发生了特异性识别事件并形成了复合物。在FFM研究中,不同表面的FFM形貌图显示出不同的表面微结构,提示抗体分子已成功固定在硫醇修饰的金衬底上,抗原修饰的探针与抗体单分子层之间存在相互作用力,封堵实验的结果与预期一致,确认了上述特异性相互作用力的存在。封堵实验结果中出现的较为“平缓”的表面结构是由于在接触模式下针尖的展宽效应所造成的。FFM成像研究与TM-AFM成像研究可互为佐证,表明两者均可用于蛋白质分子间相互作用研究,且在实验过程中发现FFM具有良好的重现性。此外,对成像过程中可能出现的假相,分别举例一一说明并给出了经验性的解决方法。(4)对抗原和抗体分子间相互作用的粘附力和摩擦力进行研究。粘附力研究结果表明抗原修饰的探针和抗体分子层间、空白探针与抗体分子层、封堵实验以及交换实验的粘附力大小分别为0.6-1.0 nN、0-0.2 nN、0-0.2 nN、1.0-1.2 nN。以上实验表明抗原与抗体间存在着特异性相互作用力。对加载速率因素的考察显示加载速率与粘附力呈两段线性关系。采用泊松分布统计法计算得到单个人IgG和大鼠抗人IgG蛋白质分子间特异性相互作用力大小为144±11 pN,非特异性相互作用为69 pN。采用FFM研究了抗原和抗体蛋白质分子间相互作用,结果表明法向加载力与摩擦力成正相关关系,抗原分子修饰的探针和抗体蛋白质分子层间、空白探针与抗体分子层、封堵实验、交换实验的摩擦力大小分别为200-250 pN、0-50 pN、50-150 pN以及250-300 pN,以上实验表明抗原与抗体间存在着特异性相互作用力,且其值比粘附力小一个数量级。上述研究结果表明,2D-GraLab是进行蛋白质分子间相互作用理论模拟的有力工具,其结果能为实验研究提供一定的指导;SAM法适用于蛋白质的固定连接,且具有可靠性和易操作性;TM-AFM和FFM成像均可用于蛋白质分子间相互作用的研究,基于AFM的粘附力和摩擦力测试揭示了蛋白质分子间相互作用的力学行为。综上所述,本文从成像与力学两个方面揭示蛋白质相互作用的分子级行为,为生物大分子相互作用研究作出了有益的探讨,并可用于生物传感器、药物筛选等生物医学重要研究领域。更多还原

【Abstract】 Proteins play essential role in biological process. Many biological functions are regulated or manipulated by protein-protein interactions. The study of interactions between antigen and antibody is one of the most improtant research fields of protein-protein interactions. In this work, two protein pairs, namely the insulin (INS)/insulin degrading enzyme (IDE) and the human IgG (antigen)/rat anti-human IgG (antibody) were selected to serve as model systems to investigate the protein-protein interactions. The research contents are as follows:(1) The interactions between INS (PDB access number: 2jv1) and IDE (PDB access number: 2jg4) were performed by a molecular simulation methods which is entitled as "Two-dimensional Graphics Lab for Biosystem Interactions". The results showed desolvation effect and Van der Waals interaction exsit during the binding process. As for the desolvation effect between INS and IDE, the desolvation free energy contributed by chain A and chain B of the complex are -4.288 kcal/mol and -5.495 kcal/mol, respectively. With respect to the Van der Waals interaction, chain A and chain B of the complex contribute -0.199 kcal/mol and -0.249 kcal/mol to it, respectively. The summarized residue-pair diagram shows there was steric clash between the Thr55 in chain A and the Thr30, and the distance between centroids is 1.71 nm. There was ion-pair interaction between the His53 in chain A and the Glu4 in chain B, and the distance between centroids is 5.35 nm.The INS interface consists of hydrophobic amino acids, thus leading to the formation of a strong hydrophobic environment in the solution of its instability, and thus as an effective chemical force driving the occurrence of combination of INS and IDE at nanomolar level. Intensive intermolecular contact had taken place in the interface of IDE and INS, resulting in an apparant matrix mosaic site and formed a pair of electrostatic interaction of the salt bridge. At the same time, under the collaborative drving of the shape complementarity of van der Waals and hydrophobic forces, IDE and INS formed the transient and stable complex structure, which mediates the downstream biological effects.(2) The 16-Mercaptohexadecanoic acid (MHA) film was prepared by self assembled method (SAM), the MHA then activated by 1-Ethyl-3- (Dimethy laminopropyl) Carbodiimide Hydrochloride (EDC) and N- Hydroxysulfosuccinimide (NHS). The antibody molecules were covalently linked on the activated MHA film and a well ordered antibody monolayer was fabricated.The obtained antibody monolayer was characterized by tapping-mode atomic force microscopy (TM-AFM), grazing incidence X-ray diffraction method (GIXRD), X-ray photoelectron spectroscopy (XPS) and contact angle genometry (CA) measurements, respectively. Both the 2D and 3D topographies of the bare gold, MHA film and the antibody monolayer were recorded by AFM, and they showed dissimilar nanostructures. The GIXRD 2θdegrees of the MHA film and the protein monolayer ranged from 0°to 15°, significantly smaller than that of the bare gold surface, but the MHA film and the protein monolayer displayed very different profiles and distributions of their diffraction peaks. Moreover, the spectra of binding energy measured from these different surfaces could be well fitted with either Au4f, S2p, or N1s, respectively. The Au4f spectra showed chemical shifts after exposure to MHA solution. With respect to S2p spectra, no detectable peaks above 164 eV were found. This means that no unbound thiol molecules presented on the MHA film. The contact angle of the MHA film and the protein monolayer were 18°and 12°, respectively, all being hydrophilic.In addition, the contact angles of mixed monolayers which formed by a series of molar ratio of MHA to dodecanethiol were also condected, the results showed the contact angle was linear with the molar ration of dodecanethiol, but reversely linear with the molar ration of MHA.(3) The interactions between antigen and antibody were imaged by TM-AFM and fiction force microscopy (FFM). As for the TM-AFM imaging, both the topographies and phase image of the bare gold, antibody monolayer and the antigen/antibody complexes were recorded by TM-AFM, respectively. The images of different surfaces showed different structure, indicating the antibody molecules were successfully immobilized on the thiol-modified gold surface and the complexes were formed due to the specific interaction between antigen and antibody.With respect to the FFM imaging, the topographies of the bare gold, the antibody monolayer, the antigen/antibody complexes, the blocking experiment and the reverse experiment were all recorded. Different surfaces showed different nanostructure, and quite in agreeable with prediction. This result suggested there are specific interaction between antigen and antibody. The pleatu-linked structure of blocking experiment was caused by the broadening effect of AFM tip. Notably, comparing to TM-AFM, the FFM measurements have better reproducibility. (4) The adhesive forces and friction forces between antigen and antibody were investigated. The adhesive force of antigen modified tip/antibody monolayer, bare tip/antibody monolayer, blocking experiment and reverse experiment were 0.6-1.0 nN, 0-0.2 nN, 0-0.2 nN and 1.0-1.2 nN, respectively, suggesting the specific interaction existed between antigen and antibody. The Possion statistical method was employed to determine the unbinding force of the single pair of antigen/antibody, and the unbinding forces and the nonspecific interaction forces was calculated to be 144±11 pN and 69 pN, respectively. Moreover, the possible artifacts during adhesive forces measurements were discussed, and the emprical solution were also provided.The friction forces between antigen and antibody were studied by FFM, the results showed the friction forces were linear with the applied normal forces. The friction forces of antigen modified tip/antibody monolayer, bare tip/antibody monolayer, blocking experiment and reverse experiment were 200-250 pN, 0-50 pN, 50-150 pN and 250-300 pN, respectively. The friction forces were one magnitude smaller than the adhesive forces. The results indicated specific interation forces between antigen and antibody. However, the detected large friction forces of bare gold/antibody monolayer may contribute to the material nature of AFM tip.The abovementioned results demostrated that 2D-GraLab is a powerful tool in computational simulating the protein-protein interactions, and it may provided helpful guide to the experimental studies. The SAM method is well suitable to immobilize the protein molecules. Besides, it is reliable and easy to do. Both the TM-AFM and FFM imaging are effective techniques to study the interaction between antigen and antibody. Both the adhesive forces and friction forces between antigen and antibody were revelaed by the AFM. Taking together, studies invovled the protein-protein interactions at the molecular level from the imaging and force points of view provide fundermental knowledge of biosensors, biomaterials and other biomedical research fields.更多还原