SPR技术分析生物分子相互作用的研究方法

生物分子的活性功能是通过分子之间的相互作用来实现的,了解这种相互作用的关系时生命科学的研究及揭示生命发生发展的基本机制具有着重要的意义.基于表面等离子共振(SPR)的分析分子相互作用(BIA)的技术是新型的生物传感技术,其无需标、能实时跟踪检测生物分子间结合、解离的整个过程,通过分析传感图谱获取分子相互作用的模式和动力学常数等方面的信息.SPR是研究生物分子相互作用的强有力工具,SPR技术已被广泛应用于生命科学领域的研究,并且显示出广阔的应用前景.概述了SPR技术原理、分析方法及其评述了其存在的问题.     

MST技术在生命科学中的应用进展

在生命科学领域中,生物分子间的相互作用具有非常重要的作用。通过分子间相互作用分析不仅可阐明细胞生物学事件,而且为疾病发生机制和药物发现提供基础。MST技术是一种基于检测在温度梯度中的生物分子电泳迁移率的变化而检测生物分子间结合、解离过程,获取分子间相互作用的模式和动力学常数等方面信息的新技术,是近年来发展的研究生物分子相互作用的强有力工具,已广泛应用于生命科学领域研究。本文综述了MST的技术原理、分析方法及其在生命科学领域的应用进展。     

SPR技术在免疫学研究中的应用

表面等离子共振(Surface Plasmon Resonance,SPR)技术是研究生物分子相互作用的强有力工具之一,该技术使生物分子之间相互作用的实时检测成为可能,并且灵敏度高、无需标记.通过分析传感图谱及分子相互作用的响应值获取分子相互作用的模式和动力学常数等方面的信息,并且获得的信息是能够定性和定量.SPR技术现在已广泛应用于生物、化学、免疫学研究及新药开发等领域.本文主要就SPR技术在免疫学研究中抗体活性检测、抗原表位预测等方面的应用进行了综述.     

单分子层次上相互作用自动检测

任何生命过程都与分子间相互作用有关。这些相互作用决定了生物分子间的＂交流＂方式,组成了生物过程的基本语言。Müller教授研究组发展了一种全自动＂机器人＂（一种全自动原子力显微镜）,可以通过检测细胞上的＂分子机器＂分析分子间相互作用。为了实现这样的目标,该仪器需要将不同的空间尺度联系起来：宏观尺度的悬臂利用其微观尺度的针尖与纳米尺度的蛋白质相接触,进而在亚纳米的尺度上定位与检测分子间相互作用。这项技术能够帮助人们以亚纳米尺度的分辨率定位细胞内分子机器的相互作用位置,并且观察分子间相互作用如何驱动这些分子机器行使功能。在药物筛选研究领域,该技术可以被用来检测配体以及抑制剂与蛋白质结合的位点和强度,还可以检测受体的不同功能状态。     

SPR生物传感器及其应用进展

基于表面等离子体共振 (SPR)技术的光学生物传感器是进行生物分子相互作用分析的一种先进手段。与传统的超速离心、荧光法等相比 ,它具有实时检测、无需标记、耗样最少等特点 ,在药物筛选、临床诊断、食物及环境监控和膜生物学等领域中的新兴应用日益扩大 ,并且已成为生命科学和制药研究的一种标准的生物物理学工具。综述了近几年国际上生物传感器的应用进展情况 ,并简要展望了该技术的发展和应用前景     

表面等离子体共振技术在分子生物学中的应用

表面等离子体共振(SPR)技术可以实时、原位地测定生物分子间的相互作用而无需任何标记,可以连续监测吸附和解离过程,并可以进行多组分复合物的相互作用的研究。SPR技术在DNA的复制和转录、DNA的修复、核酸与药物的作用以及肽库和抗体库的筛选等分子生物学领域的应用研究取得了令人瞩目的进展,显示了常规技术无法比拟的优越性。     

太赫兹(THz)光谱在生物大分子研究中的应用

太赫兹(THz)辐射是一种新型的远红外相干辐射源,近年来,在生物大分子研究中得到了广泛的应用,特别是在生物分子的结构和动力学特性等方面有着巨大的应用潜力.结合THz光谱的特点,介绍了利用THz光谱对蛋白质、糖类及DNA等生物大分子的探索研究,以及THz技术在测定水环境与生物分子相互作用等方面的应用.探讨了该技术在生物学领域应用中有待解决的问题及发展前景.     

磷烯纳米材料的生物毒性研究进展

磷烯,即单层黑磷（BP）,由于具有直接带隙、显著的结构和功能各向异性、高电荷载流子迁移率等,已经在生物医学、药物输送、生物传感、疾病的诊断和治疗等领域取得了很大的进展。和其他纳米材料相比,磷烯具有更优异的生物相容性和生物可降解性,在生物医药领域有很好的应用前景。虽然已有大量磷烯生物学效应的报道,但磷烯与生物大分子,如核酸、脂质、蛋白质之间相互作用的过程细节仍缺乏系统的研究。目前实验上无法观测磷烯与生物分子相互作用的动力学过程,分子模拟在获取精确动态结构方面具有独特的优势,被广泛应用于纳米材料和生物学领域。本文综述了近年来国内外利用计算机仿真和实验方法在磷烯纳米材料与蛋白质、脂质膜和DNA等生物大分子相互作用方面取得的最新研究进展,对磷烯生物毒性目前的研究进行了评述,并对未来需要解决的问题作了分析。本文将促进磷烯生物学效应的基础研究,也将推动磷烯纳米材料在生物医药领域的应用。     

香草酸与多酚氧化酶相互作用机制研究及新型指纹谱构建

生物大分子与小分子之间的相互作用机制研究是当今各个学科领域的前沿和热点,不仅有利于进一步认识大分子的结构和功能,还能进一步获得检测生物大分子或小分子的新途径．本研究将中药材特征指纹图谱应用于植物多酚氧化酶(polyphenol oxidase, PPO)与植物体内活性成分香草酸(vanillic acid, VA)的具体相互作用机制的研究,采用光谱实验法结合分子模拟技术,分析VA与PPO的相互作用机制,并构建其三维相互作用指纹谱．光谱实验结果显示,VA增强了PPO的荧光强度. 维持VA-PPO体系的相互作用力主要为疏水作用,VA与PPO的结合距离r值为2.48 nm,发生了非辐射能量转移．由光谱实验数据构建的λ-UV-F新型指纹图谱,系统地反映了活性分子VA与PPO之间相互作用特征．分子模拟结果精确显示了VA与PPO的结合位域与结合作用力,表明维持VA与PPO的相互作用力主要为疏水作用和氢键(位于氨基酸残基 Met258, His88, His109, His240, His244和His274位)．计算机模拟与光谱学实验结果一致,并成功构建了VA-PPO相互作用特征关系的新型指纹图谱．     

生物分子相互作用分析技术的一次突破

细胞生命活动的过程,无不涉及两个或多个生物分子之间相互作用,如信号传导、免疫反应、酶与底物作用等。因此,为了更好地研究生命过程,就迫切需要开发出一种能实时检测出生物分子间相互作用的系统,Phar－maca公司于90年代初就开发出了这样一种技术,即BM技术（BiornoecularInterationAnalssis）,该技术是基于表面等离子共振（Snd。PI。R。ce,简称SPR）的光物理现象［fi。在该现象中,所检测出的共振角与结合在传感片表面的生物分子的质量呈正比例关系。因此以时间对共振角（以共振单位表示）作图可记录相互作用的动态全过程,…     

Regulation of biomolecular condensate dynamics by signaling

Biomolecular condensates are mesoscopic biomolecular assemblies devoid of long range order that contribute to important cellular functions. They form reversibly, are stabilized by numerous but relatively weak intermolecular interactions, and their formation can be regulated by various cellular signals including changes in local concentration, post-translational modifications, energy-consuming processes, and biomolecular interactions. Condensates formed by liquid–liquid phase separation are initially liquid but are metastable relative to hydrogels or irreversible solids that have been associated with protein aggregation diseases and are stabilized by stronger, more permanent interactions. As a consequence of this, a series of cellular mechanisms are available to regulate not only biomolecular condensation but also the physical properties of the condensates.     

蓖麻毒素与其单克隆抗体相互作用动力学研究

表面等离子体激元共振(SPR)是一种可微量、实时、动态地监测生物分子相互作用的生物传感技术。蓖麻毒素为核糖体失活蛋白,具有很强的细胞毒性作用。通过SPR技术研究了两种抗蓖麻毒素的单克隆抗体C5、D12与蓖麻毒素相互作用的动力学,计算出两者的亲和常数分别为2.49×108mol-1·L和7.9×108mol-1·L,并对两种抗体的抗原表位进行了分析。     

Biomolecular interaction analysis for carbon nanotubes and for biocompatibility prediction

The interactions between carbon nanotubes (CNTs) and biologics have been commonly studied by various microscopy and spectroscopy methods. We tried biomolecular interaction analysis to measure the kinetic interactions between proteins and CNTs. The analysis demonstrated that wheat germ agglutinin (WGA) and other proteins have high affinity toward carboxylated CNT (f-MWCNT) but essentially no binding to normal CNT (p-MWCNT). The binding of f-MWCNT–protein showed dose dependence, and the observed kinetic constants were in the range of 10⁻⁹ to 10⁻¹¹ M with very small off-rates (10⁻³ to 10⁻⁷ s⁻¹), indicating a relatively tight and stable f-MWCNT–protein complex formation. Interestingly in hemolysis assay, p-MWCNT showed good biocompatibility, f-MWCNT caused 30% hemolysis, but WGA-coated f-MWCNT did not show hemolysis. Furthermore, the f-MWCNT–WGA complex demonstrated enhanced cytotoxicity toward cancer cells, perhaps through the glycoproteins expressed on the cells' surface. Taken together, biomolecular interaction analysis is a precise method that might be useful in evaluating the binding affinity of biologics to CNTs and in predicting biological actions.     

SPR studies of carbohydrate-lectin interactions as useful tool for screening on lectin sources

Lectins are proteins or glycoproteins from plants, animals or microorganisms, which typically bind specifically to sugar residues, e.g., located in cell walls or membranes. This reaction might change the physiology of the cell wall and influences the metabolism inside the cell. Some lectins of plants stimulate the immune system by unspecific activation of T-cells or influence cell division; others cause agglutination of cells (e.g., erythrocytes) and are therefore from therapeutic interest.

In a new approach, biomolecular interaction analysis (BIA) was utilised for a screening program on lectins. The BIA has been done by surface plasmon resonance (SPR). The system can be used either for characterisation of lectin-binding domains or for a screening on lectins from natural sources. Several lectin-binding surfaces on the basis of SPR have been established.     

Biophysical studies of phase separation integrating experimental and computational methods

Biomolecular phase separation that contributes to the formation of membraneless organelles and biomolecular condensates has recently gained tremendous attention because of the importance of these assemblies in physiology, disease, and engineering applications. Understanding and directing biomolecular phase separation requires a multiscale view of the biophysical properties of these phases. Yet, many classic tools to characterize biomolecular properties do not apply in these condensed phases. Here, we discuss insights obtained from spectroscopic methods, in particular nuclear magnetic resonance and optical spectroscopy, in understanding the molecular and atomic interactions that underlie the formation of protein-rich condensates. We also review approaches closely coupling nuclear magnetic resonance data with computational methods especially coarse-grained and all-atom molecular simulations, which provide insight into molecular features of phase separation. Finally, we point to future methodolical developments, particularly visualizing biophysical properties of condensates in cells.     

Optimal specificity and function for flexible biomolecular recognition

Biomolecular associations often accompanied by large conformational changes, sometimes folding and unfolding. By exploring an exactly solvable model, we constructed the free energy landscape and established a general framework for studying the biomolecular flexible binding process. We derived an optimal criterion for the specificity and function for flexible biomolecular binding where the binding and conformational folding are coupled.     

BIND: the Biomolecular Interaction Network Database

The Biomolecular Interaction Network Database (BIND: http://bind.ca) archives biomolecular interaction, complex and pathway information. A web-based system is available to query, view and submit records. BIND continues to grow with the addition of individual submissions as well as interaction data from the PDB and a number of large-scale interaction and complex mapping experiments using yeast two hybrid, mass spectrometry, genetic interactions and phage display. We have developed a new graphical analysis tool that provides users with a view of the domain composition of proteins in interaction and complex records to help relate functional domains to protein interactions. An interaction network clustering tool has also been developed to help focus on regions of interest. Continued input from users has helped further mature the BIND data specification, which now includes the ability to store detailed information about genetic interactions. The BIND data specification is available as ASN.1 and XML DTD.     

Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin

Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm “dynamics govern specificity” might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design.     

Design and synthesis of biotinylated Hexylselen as a probe to identify KGA allosteric inhibitors by a convenient biomolecular interaction assay

Hexylselen is a novel submicromolar dual KGA/GDH inhibitor, which demonstrates potent inhibition of cancer cells with minimal toxicity. To further investigation its mechanism of action, we designed and synthesized its biotinylated derivative 2 as a novel probe. From commercially available starting material, 2 was obtained in 6 steps with 13.4% overall yield. It is notable that this practical synthetic route give a template for the preparation of unsymmetrical di-benzo[d][1,2]selenazol-3(2H)-ones. Based on probe 2, we developed a novel biomolecular interaction assay for convenient and reliable test of KGA allosteric inhibitors and confirmed that hexylselen as an allosteric inhibitor of KGA sharing the same binding pocket with BPTES but not with Ebselen via competitive experiments.