【作者】 王楠；

【导师】 王远亮；

【作者基本信息】 重庆大学 ， 生物医学工程， 2004， 博士

【摘要】 蛋白质的电子传递对于生命科学的研究和新的生物分子电子器件的开发都非常重要,生命体本身就是无数酶催化反应的综合表现,大多数酶都是电子传递蛋白。金属蛋白是电子传递蛋白中的一类,因其具有纳米级尺寸、电化学活性、结构稳定、易组装和较高的电阻,这些独特的内在特性使它成为新一代分子电子材料研究的优秀的代表性模型。金属蛋白在电极表面的形貌,取向和电学特性等的研究尤其具有吸引力。而研究蛋白质的力电效应更富有挑战性。如果通过力学能够调控酶的催化反应,则具有更为深远的意义。另外,蛋白质的力电效应会直接影响蛋白质分子器件的可靠性和稳定性。本文利用原子力显微镜(AFM)为手段,以电子传递模型铜蓝蛋白(azurin)为对象,在经退火处理的金基底上,研究蛋白质的形貌以及蛋白质与基底间表面的相互作用,并考察利用突变方法控制铜蓝蛋白吸附在金基底表面的方向的可行性及其力电效应。本文主要研究两种铜蓝蛋白:野生WT型和突变体K27C型。在原子级平滑的金基底表面,获得野生WT型和K27C突变体铜蓝蛋白吸附单分子层的AFM图像,在金基底上能形成致密的铜蓝蛋白单分子层,单个铜蓝蛋白分子图像清晰可辨。WT和K27C两种铜蓝蛋白在表面分布和分子高度上都存在差异。WT铜蓝蛋白的平均直径为6nm,而K27C的为8nm。WT的高度几率分布集中在2.8nm,非常接近晶体结构数据3nm。而K27C的高度几率分布集中在3.4nm,不同于WT铜蓝蛋白。表明WT铜蓝蛋白是通过表面双硫键吸附在金基底上,而K27C铜蓝蛋白突变体是以不同方向,通过在27号位置上的半光氨酸吸附在金基底上的。利用导电原子力显微镜(Conducting-AFM)将蛋白质组装在导电针尖上与导电基底相接触,来研究铜蓝蛋白的电子传递。收集在不同外力(F)作用下的电流-电压曲线(I-V曲线)来估计蛋白质分子级电阻。在-50mV-50mV的偏压范围内,I-V曲线呈线性,符合欧姆定律。蛋白质的电阻值可直接从该直线的斜率求得(例如,在F=5nN时, 电阻R=10-9Ω)。逐渐增加力(5nN< F<70 nN),可以观察到随力变化的I-V曲线,在较低的力作用下,I-V曲线比较平缓,蛋白质电阻较大;在较高的外力作用下,I-V曲线比较陡峭,蛋白质电阻较小。实验还研究了蛋白质在压缩和撤销外力过程中与形变相关的电阻变化。因为蛋白质很软,当外力增加时,蛋白质被压缩导致电阻下降。当撤销外力时,蛋白质电阻有所增加,但并不能恢复到原来的电阻值。这表明在此力的变化范围内,蛋白质的形变是塑性的而非弹性的。有理由推测,外力作用可以改变蛋白质的空间构造,从而影响其功能。为了探索铜蓝蛋白作为一种生物分子电子器件的可能性和可靠性,我们有必<WP=6>要研究它的强度包括介电强度和机械强度。在力低于2nN时,当进行正向扫描和负向扫描,偏压高于某个临界值时,能观察到介电击穿现象。其介电强度约为109V/m。基于过高的外力能导致蛋白质分子结构瓦解的事实,我们确定了蛋白质分子的机械强度。而完全压缩蛋白质或破坏蛋白质的结构,需要的力大约80nN,相应的机械强度为2GPa。同时还发现铜蓝蛋白在一定外力作用下具有整流特性。由于蛋白质的机械强度可能与蛋白质的三维空间构造直接相关,那么维持铜蓝蛋白三级结构的力大约为80nN。本文还研究了相应的蛋白质电子传递理论,通过综合讨论有机单分子层电子传递的理论方法及其最新进展,根据在导电原子力显微镜针尖和传导基底中间夹入化学吸附的蛋白质来构造金属-绝缘体-金属结,在限定的偏压范围-1~1V内,可以观察到不对称电流曲线。由于原始的Simmons模型只能预测对称的I-V特性,而实验观察到的I-V曲线是不对称的,是故本文提出了修正的Simmons模型(考虑了费米能级在两个接触界面处的不对称偏移)来描述铜蓝蛋白的I-V特性。原始的Simmons模型和修正的Simmons模型拟合对比表明:原始的Simmons模型在较高的外力作用下,与实验数据明显地偏离;而修正模型能很好的与实验数据相吻合。这表明本研究提出的修正后的新Simmons模型是合理的,修正的理论方法是可行的。另外,本文还对蛋白质的力电效应进行了理论探讨。利用修正Simmons模型拟合得到了蛋白质分子的电子隧穿势垒高度和势垒宽度随外力增加的变化趋势。势垒宽度随外力增加而减小,而势垒高度先减小后保持不变。势垒宽度的减小是因为在外力作用下,蛋白质径向尺寸的减小。而在外力作用下势垒高度的变化趋势表明开始势垒减小是因为原子堆积密度随外力增加而增加,后来势垒高度保持不变表明蛋白子分子的原子堆积密度的增加达到了极限,本文通过理论计算推导出了相应的原子紧密堆集模型的结构参数。更多还原

【Abstract】 Electron transfer (ET) in protein has attracted much attention because it is extremely important in the bioenergetics reaction pathways. Furthermore, the study on the electron transfer of protein could help investigations into the utilization of biomolecule based on electric components. Metalloprotein, with the intrinsic properties such as nanometer dimension, electrochemical activity, robust structure, ease being engineered and comparably high resistivity, is a good candidate for next generation of electronic material. Study on its topography, orientation and electrical property on the surface of electrode is very necessary and attractive. In this thesis, the atomic force microscopy is used to investigate the topography and the surface binding interaction of protein on the annealed gold. The use of mutagenesis as a method of controlling the orientation of azurin on gold surfaces was studied. Two kinds of azurin, wild type and one genetically altered azurin mutant K27C were investigated here. Wild type azurin is immobilized using a disulphide bridge to form a covalent bond with gold surfaces. The mutant, K27C, has residues at their surface converted into cysteine residues, as it is known that thiol groups form strong covalent bonds with gold. The surface coverage and height of the adsorbed protein molecules were observed using atomic force microscopy. It is found that the density of height distribution of K27C azurin is different from that of the wild type distribution, which is possibly a result of a different orientation on the surface. It is concluded that binding of K27C mutant azurin to gold surfaces occurs preferentially through surface cysteine rather than the disulphide bridge.Electron transfer through the azurin, metalloprotein molecule has also been explored using the conducting atomic force microscopy by assembling the protein on conducting tip, which is then in conduct with the conductive substrate. The I-V curves under different force loads were collected to estimate the resistance of protein at molecular level. When reliable electrical contact between electrodes and protein is achieved under a force greater than 5 nN, well-behaved current-voltage characters are revealed, and dependent on the force load. Increasing the force from 5 to 70nN, the electrical resistance of protein is measurable, and found to be decreased <WP=8>with the force. However, the resistance could not return to the original value correspondingly as the force is withdrawn, indicating the protein deformation in this situation is not elastic.To explore the possibility and reliability of azurin being a biomolecular electrical component, study on its strengths, including the dielectric strength and mechanical strength, is also necessary. Here, the strength of azurin, has been studied at real molecular level by using conducting atomic force microscopy (C-AFM). Under a force lower than 2 nN, dielectric breakdown has been observed at a bias higher than a critical value at either positive or negative polarization. The mechanical strength of protein has been elucidated on basis of the fact that the extremely high force ranging from 60 to 100 nN leads to collapse of the protein molecular structure.In this thesis the theory of electron transfer of protein was also studied. Recent progress of study on theoretical approaches on electron transfer (ET) through organic monolayer junctions has been summarized. Metal-insulator-metal junctions have been constructed by sandwiching chemisorbed protein molecules between a conducting AFM tip and the conducting substrate. Asymmetric current curves have been observed within the confined bias region from -1 to 1 V. Due to the original Simmons model only predict the symmetric I-V behaviour, a modified Simmons model, which considered the observed spectra asymmetry in terms of inequivalent Fermi level shifts of the two contacts, was proposed to describe the I-V behaviour of azurin. A comparison of the theoretical fitting with original Simmons model and the modified model showed the modified model f更多还原