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1.
用NMR技术研究蛋白质-配体相互作用   总被引:1,自引:1,他引:0  
林东海  洪晶 《波谱学杂志》2005,22(3):321-341
蛋白质-配体相互作用的研究对理解生命过程、药物设计和药物筛选具有相当重要的科学意义和巨大的经济价值. NMR是研究蛋白质-配体相互作用的最有用的技术之一,有着显著的优势. 本文综述了近年来国际上用NMR技术研究蛋白质-配体相互作用的发展状况和趋势,先介绍表征蛋白质-配体相互作用的重要参数,然后介绍如何判断蛋白质或配体与复合物的化学交换类型以及所能获得的有关蛋白质-配体相互作用的信息,最后介绍具体用于研究蛋白质-配体相互作用的若干NMR技术以及基于NMR的药物筛选技术.  相似文献   

2.
蛋白质溶液结构及动力学的核磁共振研究   总被引:2,自引:2,他引:0  
高场液相核磁共振技术作为解析高分辨率蛋白质结构的两大主要手段之一,在近二十几年的时间里得到了迅猛的发展. 一方面,随着谱仪硬件技术、核磁脉冲技术和蛋白质标记技术的不断发展,液相核磁共振技术所能够研究的蛋白质不断突破分子量的限制,可以达到几万,甚至几十万. 另一方面,液相核磁共振技术成功地应用于蛋白质分子动力学的研究中,是目前唯一能够对蛋白质多个位点同时进行动力学研究的实验方法,并且仍在不断地创新、发展和完善中. 本文从蛋白质溶液结构的解析和动力学的研究两个主要方面对液相核磁共振研究的基本方法进行简要的介绍,并结合实例介绍一些最新的研究进展.  相似文献   

3.
张正逢  杨俊 《波谱学杂志》2013,30(2):157-174
淀粉样蛋白纤维是一类纤维状的蛋白质聚集体,与多种蛋白质沉积疾病相关. 对淀粉样蛋白纤维结构的研究,有助于人们从分子水平上阐述其形成机理, 提供相关疾病预防或治疗的依据. 由于淀粉样蛋白纤维不可溶、非结晶,因此液体核磁共振和X-射线衍射等方法对这类体系的应用受限,而固体核磁共振被认为是研究这类体系最具前景的技术. 该综述介绍了固体核磁共振解析蛋白质结构的方法及其应用于淀粉样蛋白纤维体系的研究进展.  相似文献   

4.
核磁共振(NMR)是蛋白质结构解析的主要方法之一.除可获得蛋白质的高分辨结构外,NMR还可用于观测最接近生理条件的蛋白质动态构象,获得蛋白质行使生物学功能的详细机制.非天然氨基酸定点标记方法可显著减少大分子量蛋白质的信号数目,降低数据采集和分析难度,已广泛运用于蛋白质结构和功能研究.本文介绍了常用的在蛋白质中引入非天然氨基酸的实验方法,包括蛋白质化学合成法、蛋白质化学修饰法、氟代芳香族氨基酸插入和基因密码子编辑的位点特异性插入等方法,并介绍了部分应用非天然氨基酸结合NMR研究大分子量蛋白的成功案例.此外,此篇综述讨论了目前非天然氨基酸标记在蛋白质研究中的局限性及发展方向.  相似文献   

5.
固体核磁共振技术因其可实现细胞膜环境中的蛋白质结构研究而广受关注.19F元素由于灵敏度高、天然丰度高,无生物背景等优点,被广泛应用于生物核磁共振技术中.氟标固体核磁共振技术常被用于细胞膜中蛋白质的相互作用研究,如:抗菌肽与细胞膜的相互作用、聚合膜蛋白结构分析等.此篇综述介绍了常用的蛋白质氟标修饰的实验方法,总结了常用的19F生物固体核磁共振实验技术,以及介绍了应用19F固体核磁共振研究膜蛋白的成功案例.此外,此篇综述讨论了19F固体核磁共振技术在蛋白质研究中的局限性.  相似文献   

6.
蛋白质支链动力学快运动的核磁共振研究   总被引:1,自引:1,他引:0  
蛋白质的功能不仅取决于其结构,而且受到其构像及其变化的影响. 许多生物化学过程就是由于蛋白质结构的一些动力学变化而完成,如蛋白质-蛋白质,蛋白质-药物配体之间的相互作用. 因此分析蛋白质的动力学变化,就能够对其参与的生化过程进行分析. 作为动力学研究的有力工具之一,核磁共振能够分辨到原子范围内的从千秒到皮秒时间范围的运动过程,因此在动力学研究中有着不可替代的作用. 本文仅就核磁共振在蛋白质支链快运动方法(ps-ns)研究方面的进展进行总结,以期阐明核磁共振的在支链动力学研究中的发展现状.  相似文献   

7.
α-synuclein(AS)是一种和帕金森病(PD)密切相关的天然无结构蛋白,是PD标志物路易小体(LB)的主要成分.AS异常聚集和纤维化被认为是引起PD的主要原因之一,因而针对其结构、聚集和功能的研究一直是国际上的热点.核磁共振(NMR)技术能获得具有原子分辨率的蛋白质结构和动态信息,尤其适合研究其它生物物理方法难以研究的天然无结构蛋白.该综述主要讨论NMR技术结合其他技术手段在AS的结构、聚集机制、以及与生物膜、金属离子和其它蛋白质相互作用研究中的应用.这些研究结果为探索PD的发病机制提供了有益的线索.  相似文献   

8.
配体的结合与解离过程在蛋白质实现其生物学功能方面非常关键,因此对这些高度动态过程的研究变得非常重要. 尽管已有实验方法可以确定蛋白质-配体复合物的三维结构,但一般仅可获得静态图片. 随着计算机算力的快速提高以及算法的优化,分子动力学模拟在探索配体的结合与解离过程方面具有诸多优势. 然而,当系统变得足够大时,分子动力学模拟的时间和空间尺度成为了巨大的挑战. 本工作提出了一种研究配体-蛋白质结合与解离的增强采样工具,它基于配体和蛋白质之间形成的接触数来引导迭代多组独立分子动力学模拟. 在腺苷酸激酶的模拟结果中,观测到配体的结合和解离过程,而使用传统分子动力学模拟在同一时间尺度下则无法实现这一过程.  相似文献   

9.
大肠杆菌常被用来大量快速制备重组蛋白质.但是,在原核表达目标蛋白质时非目标蛋白质经常会意外表达.有时这些非目标蛋白质也非常有使用价值,但是最终确认这些非目标蛋白质的过程昂贵又及其耗时.基于此,该文发展了一个新的基于二维核磁共振波谱技术、X-单晶衍射技术、结合其他生物化学方法,确认在原核表达神经限制性沉默因子NRSF/REST蛋白(该蛋白能够特异性识别神经限制性沉默因子RE1ds DNA及神经限制性激活因子ds RNA,以调节神经元干细胞的发育)功能结构域Zn F2-8时非目标蛋白β-内酰胺酶(β-lactamase).  相似文献   

10.
近年来,结构生物学研究越来越注重生物大分子复合物的解析,因为许多重要生物学过程都离不开复合物的参与.溶液核磁共振是目前重要的结构解析方法之一.X射线小角散射(SAXS)作为一种新的结构生物学实验手段,近年来发展迅速.SAXS能提供生物大分子复合物的较低分辨率结构信息,而核磁共振能解析复合物中各个亚基的原子分辨率结构.此外,通过核磁共振还能得到亚基之间的界面、取向以及距离信息.因此近年来通过计算机模拟,整合核磁共振和SAXS不同分辨率的结构信息,可以用来搭建生物大分子复合物的结构模型.该综述重点介绍这方面的研究进展.  相似文献   

11.
Nuclear magnetic resonance (NMR) crystallography is an approach for revealing molecular and supramolecular structures and molecular packing for systems where standard X-ray crystallography gives no results. It combines solid-state NMR techniques with chemical models and/or molecular dynamics and/or quantum chemical calculations. These techniques are often supported by other structure characterization methods. In the present review, recent results on the application of NMR crystallography for the investigation of the mode of action of superoxide dismutases are discussed. Studies of substrate–inhibitor complexes of human manganese and Streptomyces nickel superoxide dismutase are presented, which are chemical models of the transient enzyme–substrate complex. The review is completed by new, previously unpublished results, calculating an NMR structure of NiSOD model peptide-bound cyanide based on experimental restraints measured by us and derived from the literature and extended DFT calculations.  相似文献   

12.
Molecular modelling is a powerful methodology for analysing the three dimensional structure of biological macromolecules. There are many ways in which molecular modelling methods have been used to address problems in structural biology. It is not widely appreciated that modelling methods are often an integral component of structure determination by NMR spectroscopy and X-ray crystallography. In this review we consider some of the numerous ways in which modelling can be used to interpret and rationalise experimental data and in constructing hypotheses that can be tested by experiment. Genome sequencing projects are producing a vast wealth of data describing the protein coding regions of the genome under study. However, only a minority of the protein sequences thus identified will have a clear sequence homology to a known protein. In such cases valuable three-dimensional models of the protein coding sequence can be constructed by homology modelling methods. Threading methods, which used specialised schemes to relate protein sequences to a library of known structures, have been shown to be able to identify the likely protein fold even in cases where there is no clear sequence homology. The number of protein sequences that cannot be assigned to a structural class by homology or threading methods, simply because they belong to a previously unidentified protein folding class, will decrease in the future as collaborative efforts in systematic structure determination begin to develop. For this reason, modelling methods are likely to become increasingly useful in the near future. The role of the blind prediction contests, such as the Critical Assessment of techniques for protein Structure Prediction (CASP), will be briefly discussed. Methods for modelling protein-ligand and protein-protein complexes are also described and examples of their applications given.  相似文献   

13.
The fluorescence properties of protopine and allocryptopine in aqueous and organic environments are described for the first time. The fluorescence of alkaloids and their pH-dependent interconversion to cationic forms (transannular interaction) were studied using steady-state and time-resolved fluorescence techniques. For the analysis of tricyclic base and cis/trans tetracyclic cations of the alkaloids, NMR and X-ray crystallography were used.  相似文献   

14.
In this paper, we showed the application of solid state-NMR (SS NMR) spectroscopy in structural studies of chiral compounds employing sample of (E)-1-diphenylphosphinoylpent-3-en-2-ol 1 as a model compound. Racemate of 1 was fully characterized by NMR techniques (both in liquid and solid phase) and X-ray crystallography. Theoretical calculations employing the GIAO approach were used to explain the influence of hydrogen bonding on 31P NMR shielding parameter in racemate. Enantioselective inclusion complexation (EIC) method with TADDOL as host molecule was applied to separate of enantiomers. The formation of host-guest complex and decomplexation procedure was monitored by means of the SS NMR. The liquid-state NMR, due to similarity of 13C and 31P spectral parameters was not able to distinguish racemate from enantiomer. In the solid phase, owing to distinction of hydrogen bonding and molecular packing in the crystal lattice, racemate and enantiomers were easy recognized by NMR spectroscopy.  相似文献   

15.
16.
用核磁共振方法研究金属离子与蛋白质的相互作用   总被引:2,自引:1,他引:1  
张芳  林东海 《波谱学杂志》2009,26(1):136-149
许多蛋白质含有金属离子,金属离子对蛋白质发挥生物学功能起着很大的作用. 金属离子与蛋白质的相互作用以及参与蛋白质功能调节的方式各种各样:有些金属离子高度专一性地与蛋白质紧密结合,对蛋白质发挥生物学功能起着关键性的作用;有些金属离子只是作为蛋白质发挥功能的辅助因子而瞬态地与蛋白质松散结合. 本文简要介绍目前国际上用NMR方法研究抗磁金属离子和顺磁金属离子与蛋白质相互作用的进展,并具体介绍了NMR方法在钙调蛋白、锌指蛋白、朊病毒蛋白等金属离子蛋白研究上的应用.  相似文献   

17.
Molluscan hemocyanins are proteins of a truly enormous size. Because of this, determination of their quaternary structure at high resolution cannot easily be obtained by standard methods such as X-ray crystallography and NMR. Therefore, different approaches, using several low-resolution techniques are currently necessary to understand hemocyanin structure. In this work a model of the Rapana venosa hemocyanin has been obtained from a template model and small angle X-ray scattering (SAXS) data. The template model was built from the electron density of the closely related Haliotis tuberculata hemocyanin and a computer program was written to fit this model to the SAXS data using the simulated annealing algorithm.  相似文献   

18.
The ENDOR crystallography of disordered systems used for studying the electronic and geometric structure of some copper(II) sulfur containing chelate complexes and oxovanadium(II) chelate complexes in solid state and in frozen solution is reported. This method is also applied for studying some very weak interactions in solution between the cited chelate complexes and different solvents (alcohol, chloroform) which are unaccessible with other physical methods.  相似文献   

19.
Three different methods are currently used for the study of high-resolution structures of membrane proteins: X-ray crystallography, electron crystallography, and nuclear magnetic resonance (NMR) spectroscopy. Thus far, all methods combined have yielded a rather modest number of crystal structures that have been solved at the atomic level. It is hypothesized here that different methods may select different populations of proteins on the basis of various properties. Thus, protein stability may be a significant factor in the formation of three-dimensional (3D) crystals from detergent solutions, since exposure of hydrophobic protein zones to water may cause structural perturbation or denaturation in conformationally labile proteins. This is different in the formation of two-dimensional (2D) crystals where a protein remains protected in its native membrane environment. A biological selection mechanism may therefore be operative in that highly ordered lattices may form only if strong protein-protein interactions are relevant in vivo, thereby limiting the number of proteins that are amenable to electron crystallography. Keeping a protein in a bilayer environment throughout 3D crystallization maintains the lateral pressure existing in native membranes. This can be accomplished by using lipidic cubic phases. Alternatively, the hydrophobic interface of a membrane protein may be spared from contact with water by crystallization from organic solvents where the polar caps are protected in reverse micelles by using appropriate detergents. Some of the criteria that are useful in optimizing the various approaches are given. While the usefulness of complementary methods seems obvious, the results presented may be particularly critical in recognizing key problems in other structural approaches.  相似文献   

20.
近年来,结构生物学研究越来越注重生物大分子复合物的解析,因为许多重要生物学过程都离不开复合物的参与.溶液核磁共振是目前重要的结构解析方法之一.X射线小角散射(SAXS)作为一种新的结构生物学实验手段,近年来发展迅速.SAXS 能提供生物大分子复合物的较低分辨率结构信息,而核磁共振能解析复合物中各个亚基的原子分辨率结构.此外,通过核磁共振还能得到亚基之间的界面、取向以及距离信息.因此近年来通过计算机模拟,整合核磁共振和 SAXS 不同分辨率的结构信息,可以用来搭建生物大分子复合物的结构模型.该综述重点介绍这方面的研究进展.  相似文献   

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