血红蛋白在自组装单层修饰电极上的直接电化学研究

对于氧化还原蛋白质在电极界面上的直接电子转移的研究对于生物传感器的构建,生物材料的发展和生物系统的认识有很重要的作用~([1]).虽然许多方法已经实现了蛋白质的直接电子传递,这些方法多数侧重于生物传感器的构建,对如何通过控制蛋白质的吸附状态来实现直接电子传递并没有系统的报道.蛋白质在吸附到电极表面的过程中,许多因素都能影响蛋白质的电子传递,如蛋白质的取向,蛋白质的活性中心与电极的距离等.     

界面电场对血红蛋白在多孔金电极上的吸附和生物活性的影响(英文)

蛋白质的界面吸附及其生物活性因它在构建生物传感、生物电子器件和生物燃料电池等方面具有重要的作用而倍受关注.对此,界面电场是吸附的一个重要影响因素,它能明显地影响蛋白质分子在材料界面的吸附量、分子构象以及分子定向.本文应用电化学方法和红外光谱技术研究了血红蛋白在三维多孔金膜电极上的吸附动力学及其生物活性随界面电场的变化关系.结果表明,由界面电场产生的过量表面电荷可借助与蛋白质分子之间的静电作用加速蛋白质分子在电极表面的吸附,提高其吸附量;但是,过高的界面电场将破坏吸附蛋白质的构象以及降低它还原过氧化氢的催化活性;只有在零电荷电位下,吸附在电极表面的血红蛋白才能保持其天然的构象和生物催化活性.本研究将为生物传感器、生物电子器件和生物燃料电池的构建提供理论依据,加深对荷电生物界面上生物分子界面行为的认识.     

BSA和SDBS在水溶液与空气界面的相互作用

表面活性剂与蛋白质在界面处的相互作用在许多应用里都会遇到．如去污、药品或生物制剂的制备和提纯、某些开科手术等等[1]这常常涉及到表面活性剂与蛋白质在界面的吸附，蛋白质大分子在界面吸附层的取向和构象．对于这两种物质在界面处可能发生的相互作用的性质，是表面活性剂     

蛋白质相互作用: 界面分析, 结合自由能计算与相互作用设计

蛋白质相互作用在生命活动中起着重要作用. 研究蛋白质间相互作用的本质有助于了解生命活动中这些基本单元的作用. 本文主要综述了近期蛋白质相互作用研究的进展, 包括蛋白质相互作用界面的基本性质, 蛋白质结合自由能的计算方法, 不同相互作用在蛋白质结合/解离中的角色和差异, 以及上述知识在蛋白质相互作用设计中的应用. 蛋白质相互作用界面的特性, 例如界面大小、保守性以及结构的动态性质, 使得具有生物功能的蛋白质相互作用界面区别于非特异性的晶体堆积界面. 生物功能界面的一个重要结构特征是界面上存在着关键残基以及相对独立的相互作用模块. 利用多种方法, 如MM-PBSA、统计平均势以及不同的相互作用自由能模型, 可以在不同的精度上计算蛋白质相互作用自由能. 利用蛋白质相互作用界面的特点, 从不同的角度进行蛋白质相互作用对的设计与改造, 近年来已经有了不少成功的例子, 但还存在着很大的挑战. 我们认为在今后的蛋白质相互作用设计中, 考虑各种因素对蛋白质结合与解离的动力学过程的影响将有助于提高人类控制蛋白质相互作用的能力.     

无机纳米-生物界面作用的SERS研究

探索生命体对无机纳米材料的生物应答机制是高效、安全、可控地应用无机纳米材料的基础,其关键在于准确理解在生物体系中无机纳米材料与生物分子间的纳米.生物界面作用.本文主要探讨了在纳米-生物界面具有拉曼增强效应的金、银纳米材料;介绍了表面增强拉曼光谱(surface-enhanced raman spectroscopy,SERS)原位研究金、银等无机纳米材料表/界面吸附的核酸、蛋白质、磷脂等生物分子,以及细胞、病毒和细菌等与金、银纳米材料表/界面作用的研究进展;综述了SERS技术在探索纳米-生物界面作用机制、生物分子测定、生物分子界面行为监测中的应用.     

鳞状上皮细胞癌抗原2与组蛋白G的柔性对接研究

蛋白质与蛋白质间的相互作用在生物体内具有极其重要的意义 .确定蛋白质间的相互作用的机制对生物调节和药物设计都是关键的步骤 .近年来 ,随着计算机和软件技术的发展 ,使建立蛋白质 -蛋白质复合物模型成为可能 ,为研究蛋白质间的相互作用提供了直观和比较准确的参考 .目前的蛋白质 -蛋白质对接软件将对接的两个蛋白质看成刚性 ,并不考虑蛋白质对接时的柔性 ,即没有考虑由于界面氨基酸间的相互作用而导致的界面结构的变化 .本文在直接的蛋白质 -蛋白质刚性对接的基础上 ,应用动力学模拟的方法 ,对刚性对接的结构进行蛋白质界面的构象搜索 ,…     

生物材料表界面与蛋白质及细胞的相互作用的研究进展

材料接触生物环境首先是通过其界面,这种材料界面与生物环境中的生物分子及细胞之间的相互作用决定着材料生物功能的实现。因此,调控材料与生物体的界面相互作用几乎是所有生物材料的研究及应用首先和必然面对的关键共性问题。本文综述了近年来我们课题组在生物表界面领域的研究及其最新进展。从分子层面上设计生物功能表面入手,建立了一系列普适、高效、简单易行的表面功能化新方法用于改变材料表面的物理化学性质,进而调控材料表面与蛋白质或细胞/细菌之间的相互作用。     

无机纳米晶-生物界面作用的分子机制

无机纳米晶材料以其独特的光、电、磁、力学性质,成为疾病诊断与治疗功能的关键材料.本文总结了无机纳米晶的表面化学活性、离子释放性、晶相结构、晶格缺陷、表面吸附和表面修饰等与尺寸相关的理化性质与生物效应之间的关系.综述了无机纳米晶与蛋白质、磷脂生物膜间的界面相互作用,探讨了纳米晶-生物界面作用的分子机理.这有助于理解无机纳米晶的生物行为和毒理性质,指导设计安全、高效的纳米晶生物医学材料.     

PNIPAAm改性表面对蛋白质吸附的调控及其应用

根据不同领域的需要,控制蛋白质在材料表面的吸附是一个具有重要应用价值的课题。聚（N-异丙基丙烯酰胺）（PNIPAAm）改性表面能够响应外界温度变化从而改变其表面性质,这一特点为调控蛋白质的吸附提供了可能。近年来,研究者们应用多种表征方法考察了不同温度下蛋白质在PNIPAAm改性表面的吸附,并试图从分子水平上深入理解其吸附机制及影响因素。本文综述了近年来应用PNIPAAm改性表面对蛋白质吸附的研究及其最新进展。发现当PNIPAAm层厚度处于一定范围内时,PNIPAAm改性表面表现出对蛋白质吸附的温度敏感性,并可以利用这一性质将其应用于蛋白质纯化及分离和生物传感器等领域。而当PNIPAAm层厚度超过一个临界值时,PNIPAAm改性表面表现出良好的阻抗血浆蛋白质的性质,使其有望在血液相容性表面领域得到应用。最后,就PNIPAAm改性表面调控蛋白质吸附的未来发展方向简要地进行了展望。     

抗污界面构建及其电化学生物传感应用进展

电化学生物传感器具有灵敏度高、便携性好、响应快速和易于集成等优点,在临床检测方面有很大应用潜力,并在可穿戴健康监测领域得到了快速发展。但在实际临床生物样本检测中,非靶标生物物质会在电极表面产生非特异性吸附（即生物污染）,影响了电化学生物传感器的性能。因此,构建具有防污染能力的传感界面（抗污界面）,防止非靶标物质吸附到电极表面,对于扩大电化学生物传感器的实际应用范围,实现在复杂生物样本中的检测至关重要。本文概述了物理、化学和生物抗污电极界面的构建及其在临床相关生物标志物检测中的应用,为电化学生物传感器实际应用性能的提升提供技术参考,并通过对界面抗污原理和存在问题的探讨,对抗污界面发展前景和未来趋势予以展望。     

Molecular packing of lysozyme,fibrinogen, and bovine serum albumin on hydrophilic and hydrophobic surfaces studied by infrared-visible sum frequency generation and fluorescence microscopy

Infrared-visible sum frequency generation (SFG) vibrational spectroscopy, in combination with fluorescence microscopy, was employed to investigate the surface structure of lysozyme, fibrinogen, and bovine serum albumin (BSA) adsorbed on hydrophilic silica and hydrophobic polystyrene as a function of protein concentration. Fluorescence microscopy shows that the relative amounts of protein adsorbed on hydrophilic and hydrophobic surfaces increase in proportion with the concentration of protein solutions. For a given bulk protein concentration, a larger amount of protein is adsorbed on hydrophobic polystyrene surfaces compared to hydrophilic silica surfaces. While lysozyme molecules adsorbed on silica surfaces yield relatively similar SFG spectra, regardless of the surface concentration, SFG spectra of fibrinogen and BSA adsorbed on silica surfaces exhibit concentration-dependent signal intensities and peak shapes. Quantitative SFG data analysis reveals that methyl groups in lysozyme adsorbed on hydrophilic surfaces show a concentration-independent orientation. However, methyl groups in BSA and fibrinogen become less tilted with respect to the surface normal with increasing protein concentration at the surface. On hydrophobic polystyrene surfaces, all proteins yield similar SFG spectra, which are different from those on hydrophilic surfaces. Although more protein molecules are present on hydrophobic surfaces, lower SFG signal intensity is observed, indicating that methyl groups in adsorbed proteins are more randomly oriented as compared to those on hydrophilic surfaces. SFG data also shows that the orientation and ordering of phenyl rings in the polystyrene surface is affected by protein adsorption, depending on the amount and type of proteins.     

Imaging the binding ability of proteins immobilized on surfaces with different orientations by using liquid crystals

We report an investigation of the binding ability of a protein immobilized on surfaces with different orientations but in identical interfacial microenvironments. The surfaces present mixed self-assembled monolayers (SAMs) of 11-[19-carboxymethylhexa(ethylene glycol)]undecyl-1-thiol, 1, and 11-tetra(ethylene glycol) undecyl-1-thiol, 2. Whereas 2 is used to define an interfacial microenvironment that prevents nonspecific adsorption of proteins, 1 was activated by two different schemes to immobilize ribonuclease A (RNase A) in either a preferred orientation or random orientations. The binding of the ribonuclease inhibitor protein (RI) to RNase A on these surfaces was characterized by using ellipsometry and the orientational behavior of liquid crystals. Ellipsometric measurements indicate identical extents of immobilization of RNase A via the two schemes. Following incubation of both surfaces with RI, however, ellipsometric measurements indicate a 4-fold higher binding ability of the RNase A immobilized with a preferred orientation over RNase A immobilized with a random orientation. The higher binding ability of the oriented RNase A over the randomly oriented RNase A was also apparent in the orientational behavior of nematic liquid crystals of 4-cyano-4'-pentylcyanobiphenyl (5CB) overlayed on these surfaces. These results demonstrate that the orientations of proteins covalently immobilized in controlled interfacial microenvironments can influence the binding activities of the immobilized proteins. Results reported in this article also demonstrate that the orientational states of proteins immobilized at surfaces can be distinguished by examining the optical appearances of liquid crystals.     

Multiscale modeling and simulations of protein adsorption: progresses and perspectives

Protein adsorption, which shows wide prospects in many practical applications such as biosensors, biofuel cells, and biomaterials, has long been identified as a very complex problem in interface science. Here, we present a review on the multiscale modeling and simulation methods of protein adsorption on surfaces with different properties. First, various simulation algorithms (replica exchange, metadynamics, TIGER2A, and PSOVina) and protein models (colloidal, coarse-grained, and all-atom models) are introduced. Then, recent molecular simulation progresses about protein adsorption on different material surfaces (such as charged, hydrophobic, hydrophilic, and responsive surfaces) are retrospected. It has been demonstrated that the adsorption orientation of proteins on charged surfaces and hydrophobic surfaces can be controlled by the electrical dipole and the hydrophobic dipole of proteins, respectively. Superhydrophilic zwitterionic surfaces can resist protein adsorption because of the strong hydration. Under the stimuli of external conditions, the surface properties of materials can be modulated, and thus, the adsorption/desorption of proteins on responsive surfaces can be controlled. Finally, the future directions of molecular simulation study of protein adsorption are discussed.     

Oriented protein adsorption to gold nanoparticles through a genetically encodable binding motif

Simple, stable, and specific methods for immobilizing proteins on gold surfaces are needed for the development of applications that rely on the oriented attachment of proteins to gold surfaces. We report a direct, stable, genetically encodable method for the oriented chemisorption of proteins to gold nanoparticles (Au NPs) through the tetracysteine motif (C-C-P-G-C-C) while simultaneously suppressing protein physisorption. Mutants of ubiquitin (Ub) and enhanced green fluorescent protein (eGFP) containing the tetracysteine motif were produced and displayed stronger adsorption to the NPs than did native proteins. An eGFP mutant with a dicysteine motif (G-C-C) did not show a significant improvement in binding to Au NPs compared to that of the wild-type protein. The binding of the proteins to Au NPs of various sizes (14, 18, 28, and 39 nm) was explored. The small Ub tetracysteine mutant stabilized several sizes of Au NPs, and the eGFP tetracysteine mutant clearly had the strongest chemisorption to the 18 nm NPs. The control of binding orientation for proteins bearing a tetracysteine motif was demonstrated through the enhanced specific binding of protein-NP conjugates to immobilized targets.     

A genetic approach for controlling the binding and orientation of proteins on nanoparticles

Although silver nanoparticles are excellent surface enhancers for Raman spectroscopy, their use to probe the conformation of large proteins at interfaces has been complicated by the fact that many polypeptides adsorb weakly or with a random orientation to colloidal silver. To address these limitations, we sought to increase binding affinity and control protein orientation by fusing a silver-binding dodecapeptide termed Ag4 to the C-terminus of maltose-binding protein (MBP), a well-characterized model protein with little intrinsic silver binding affinity. Quartz crystal microbalance measurements conducted with the MBP-Ag4 fusion protein revealed that its affinity for silver (Kd approximately 180 nM) was at least 1 order of magnitude higher than a control protein, MBP2, containing a non-silver-specific C-terminal extension. Under our experimental conditions, MBP-Ag4 SERS spectra exhibited 2-4 fold higher signal-to-background relative to MPB2 and contained a number of amino acid-assigned vibrational modes that were either weak or absent in control experiments performed with MBP2. Changes in amino acid-assigned peaks before and after MBP-Ag4 bound maltose were used to assess protein orientation on the surface of silver nanoparticles. The genetic route described here may prove useful to study the orientation of other proteins on a variety of SERS-active surfaces, to improve biosensors performance, and to control functional nanobiomaterials assembly.     

Viscoelastic modeling of highly hydrated laminin layers at homogeneous and nanostructured surfaces: quantification of protein layer properties using QCM-D and SPR

The adsorption of proteins at material surfaces is important in applications such as biomaterials, drug delivery, and diagnostics. The interaction of cells with artificial surfaces is mediated through adsorbed proteins, where the type of protein, amount, orientation, and conformation are of consequence for the cell response. Laminin, an important cell adhesive protein that is central in developmental biology, is studied by a combination of quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance (SPR) to characterize the adsorption of laminin on surfaces of different surface chemistries. The combination of these two techniques allows for the determination of the thickness and effective density of the protein layer as well as the adsorbed mass and viscoelastic properties. We also evaluate the capacity of QCM-D to be used as a quantitative technique on a nanostructured surface, where protein is adsorbed specifically in a nanopattern exploiting PLL-g-PEG as a protein-resistant background. We show that laminin forms a highly hydrated protein layer with different characteristics depending on the underlying substrate. Using a combination of QCM-D and atomic force microscopy (AFM) data from nanostructured surfaces, we model laminin and antibody binding to nanometer-scale patches. A higher amount of laminin was found to adsorb in a thicker layer of a lower effective density in nanopatches compared to equivalent homogeneous surfaces. These results suggest that modeling of QCM-D data of soft viscoelastic layers arranged in nanopatterns may be applied where an independent measure of the "dry" mass is known.     

Nanoporous microbead supported bilayers: stability, physical characterization, and incorporation of functional transmembrane proteins

The introduction of functional transmembrane proteins into supported bilayer-based biomimetic systems presents a significant challenge for biophysics. Among the various methods for producing supported bilayers, liposomal fusion offers a versatile method for the introduction of membrane proteins into supported bilayers on a variety of substrates. In this study, the properties of protein containing unilamellar phosphocholine lipid bilayers on nanoporous silica microspheres are investigated. The effects of the silica substrate, pore structure, and the substrate curvature on the stability of the membrane and the functionality of the membrane protein are determined. Supported bilayers on porous silica microspheres show a significant increase in surface area on surfaces with structures in excess of 10 nm as well as an overall decrease in stability resulting from increasing pore size and curvature. Comparison of the liposomal and detergent-mediated introduction of purified bacteriorhodopsin (bR) and the human type 3 serotonin receptor (5HT3R) are investigated focusing on the resulting protein function, diffusion, orientation, and incorporation efficiency. In both cases, functional proteins are observed; however, the reconstitution efficiency and orientation selectivity are significantly enhanced through detergent-mediated protein reconstitution. The results of these experiments provide a basis for bulk ionic and fluorescent dye-based compartmentalization assays as well as single-molecule optical and single-channel electrochemical interrogation of transmembrane proteins in a biomimetic platform.     

Surface tailoring for controlled protein adsorption: effect of topography at the nanometer scale and chemistry

Protein adsorption behavior is at the heart of many of today's research fields including biotechnology and materials science. With understanding of protein-surface interactions, control over the conformation and orientation of immobilized species may ultimately allow tailor-made surfaces to be generated. In this contribution protein-surface interactions have been examined with particular focus on surface curvature with and without surface chemistry effects. Silica spheres with diameters in the range 15-165 nm with both hydrophilic and hydrophobic surface chemistries have been used as model substrates. Two proteins differing in size and shape, bovine serum albumin (BSA) and bovine fibrinogen (Fg), have been used in model studies of protein binding with detailed secondary structure analysis being performed using infrared spectroscopy (IR) on surface-bound proteins. Although trends in binding affinity and saturation values were similar for both proteins, albumin is increasingly less ordered on larger substrates, while fibrinogen, in contrast, loses secondary structure to a greater extent when adsorbing onto particles with high surface curvature. These effects are compounded by surface chemistry, with both proteins becoming more denatured on hydrophobic surfaces. Both surface chemistry and topography play key roles in determining the structure of the bound proteins. A model of the binding characteristics of these two proteins onto surfaces having differing curvature and chemistry is presented. We propose that properties of an adsorbed protein layer may be guided through careful consideration of surface structure, allowing the fabrication of materials/surface coatings with tailored bioactivity.     

Characterization of protein immobilization at silver surfaces by near edge X-ray absorption fine structure spectroscopy

Ribonuclease A (RNase A) is immobilized on silver surfaces in oriented and random form via self-assembled monolayers (SAMs) of alkanethiols. The immobilization process is characterized step-by-step using chemically selective near-edge X-ray absorption fine structure spectroscopy (NEXAFS) at the C, N, and S K-edges. Causes of imperfect immobilization are pinpointed, such as oxidation and partial desorption of the alkanethiol SAMs and incomplete coverage. The orientation of the protein layer manifests itself in an 18% polarization dependence of the NEXAFS signal from the N 1s to pi* transition of the peptide bond, which is not seen for a random orientation. The S 1s to C-S sigma* transition exhibits an even larger polarization dependence of 41%, which is reduced to 5% for a random orientation. A quantitative model is developed that explains the sign and magnitude of the polarization dependence at both edges. The results demonstrate that NEXAFS is able to characterize surface reactions during the immobilization of proteins and to provide insight into their orientations on surfaces.     

Strategies for protein-based nanofabrication: Ni-NTA as a chemical mask to control biologically imposed symmetry

Background: Technologies that improve control of protein orientation on surfaces or in solution, through designed molecular recognition, will expand the range of proteins that are useful for biosensors, molecular devices and biomaterials. A limitation of some proteins is their biologically imposed symmetry, which results in indistinguishable recognition surfaces. Here, we have explored methods for modifying the symmetry of an oligomeric protein that exhibits useful self-assembly properties.Results:Escherichia coli glutamine synthetase (GS) contains 24 solvent-exposed histidines on two symmetry-related surfaces. These histidines drive a metal-dependent self-assembly of GS tubes. Immobilization of GS on the affinity resin Ni²⁺-NTA followed by on-column modification with diethyl pyrocarbonate affords asymmetrically modified GS that self-assembles only to the extent of ‘short’ dimeric GS tubes, as demonstrated by electron microscopy, dynamic light scattering and atomic force microscopy. The utility of Ni²⁺-NTA as a chemical mask was also demonstrated for asymmetric modification of engineered cysteines adjacent to the natural histidines.Conclusions: Current genetic methods do not provide distinguishable recognition elements on symmetry-related surfaces of biologically assembled proteins. Ni²⁺-NTA serves as a mask to control chemical modification in vitro of residues within symmetry-related pairs, on proteins containing functional Histags. This strategy may be extended to modification of a wide range of amino acids with a myriad of reagents.