核磁共振技术及其在生命科学中的应用

核磁共振(NMR)是一种不破坏样品的无损伤分析技术,是分子结构分析不可或缺的手段。随着新技术、新方法的不断发展,其研究领域和应用范围已扩展到了几乎所有的自然科学领域。本文简要介绍了核磁共振波谱技术的基本原理、多维NMR以及在结构鉴定、构象分析、医学临床检测、蛋白质组学、代谢组学等方面的应用。同时,本文还就NMR技术的发展前景提出了一些看法。     

核磁共振技术在药物检测中的应用进展CSCD

核磁共振(nuclear magnetic resonance, NMR)技术已发展成为新化合物结构鉴定和天然产物药物分析不可缺少的工具。因其具有非破坏性和无侵入性等特点,广泛应用于药物领域,从合成产物的表征到生物系统分子结构和构象的测定以及分析分子间相互作用等研究都离不开这个工具。核磁共振能提供丰富且精确的结构信息,如拉莫尔频率、化学位移、自旋-自旋偶合、偶极偶合和弛豫时间等参数,成为结构解析的“金标准”。本文总结了核磁共振技术在药物发现、药物相互作用、药物代谢组学以及固态核磁共振技术在固态药物分析中的应用进展。     

核磁共振技术在生物物理学领域中的应用

核磁共振波谱法(NMR)是利用核磁共振波谱对纯化合物进行结构测定,并定性或定量分析其微结构的一种研究手段.随着脉冲傅立叶变换技术和超导磁体的发展和普及.15N和31P等核磁共振谱(以15N和31P原子为测定对象得出的图谱),以及诸如二维核磁共振谱等新技术和新方法不断涌现并完善,核磁共振波谱法在生物学领域的应用也越来越广泛.在调研大量国外文献基础上,介绍NMR方法在生命科学研究方面的应用研究成果.     

核磁共振技术在蛋白质领域的应用

简要叙述了核磁共振技术(NMR)在蛋白质领域的研究及应用。NMR法通过测定蛋白质在稀溶液状态下反应位点的特定参数来计算蛋白质的三级结构,并可深入了解一定时间范围内化学反应和蛋白质构象转变的动力学过程。通过NMR对抗原决定簇和抗体CDR作图,可以分析其一级结构和三维构象;对抗原抗体动力学的分析,对于设计基因疫苗、检测细胞表面抗原提呈以及分析抗原抗体复合物的构象变化也有着重要意义。     

核磁共振技术在土壤-植物-大气连续体研究中的应用

植物体内的水分状态与传输过程是土壤-植物-大气连续体(SPAC)水分传输理论的核心内容,也是研究植物水分利用与调控的基础.植物体内水分的传输过程受外界环境影响较大,植物需要通过对体内水分状态的适当调整来适应环境变化和维持自身的生长发育.由于蒸发通量、压力室、高压流速仪、热脉冲等传统检测方法往往会对植株造成破坏和损伤,因此难以准确反映和定量描述植物体内水分传输的真实过程.核磁共振技术(NMR)由于其无损、非侵入的特点,在植物水分分布和传输相关研究中日益得到关注.本文概述了NMR在检测植物体内水分分布、传输以及含量测定等方面的研究进展,还分析了目前NMR技术在SPAC系统研究中存在的问题及可能的解决方法,并指出NMR技术将来可能在植物水分生理、植物与环境互作以及水分代谢等相关研究领域的应用.NMR技术在SPAC系统研究中的应用在我国仍处于初级阶段,开发户外便携式、开放式检测仪器是NMR技术在SPAC研究领域进一步应用和推广的关键所在.     

二维核磁共振谱在多糖结构研究中的应用

二维核磁共振谱（2D NMR）是获取多糖结构信息,尤其是在多糖序列分析方面的有力工具。本文重点介绍了在多糖结构解析中常用的几种2D NMR谱以及2D NMR解析多糖结构的方法。     

一种百合甾体皂苷纯度及糖链结构核磁共振法分析

采用多种NMR分析技术,首次对百合甾体皂苷(25R,26R)-26-甲氧基螺甾烷-5-烯-3β-α-L-鼠李糖-(1→2)-[β-D-葡萄糖-(1→6)]-β-D-葡萄糖苷的1H和13C NMR信号进行了全归属,特别是应用选择性的1D TOCSY和1D NOESY核磁共振分析技术,对该化合物1中的氢谱信号严重重叠的糖链进行了详细的分析,提出了一套对甾体皂苷糖链信号进行全归属的核磁共振法.在确认其结构的基础上,建立了核磁共振法(1H NMR)测定该化合物1的纯度,给出了完整的实验条件,线性回归系数为0.9998,重复性实验RSD为0.58％,稳定性实验RSD为0.24％,操作简单、快速准确,且不需要其它对照品,是中药化学对照品纯度研究的一个有益补充.     

代谢组学与营养学研究

代谢组学(metabonomics)是近几年来基于核磁共振(nuclear magnetic resonance,NMR)、质谱(mass spectrometry,MS)和高效液相色谱(HPLC)等发展起来的一种新的"组学"技术,是通过分析生物体液及组织中所有小分子物质来研究有机体内物质代谢规律和健康状况的一门学科。代谢组学在功能基因组学、病理生理学、药理毒理学和营养学等方面有着广泛的应用前景。本文综述了代谢组学的研究进展和在营养学方面的应用。     

核磁共振用于肿瘤研究的进展

近代自然科学发展的一个重要特点是数学、物理学等精密学科以及工程技术向生物科学的渗透,用来阐明生命过程的细微机理,并力图寻找其在医学、农业等方面的实际意义。核磁共振术在肿瘤研究中的应用就是一个明显的实例。1971年,Damadian等首次把核磁共振(以下简称为NMR)技术用于研究正常及接种肿瘤的大鼠组织,发现癌组织与正常组织的一些NMR参量有明显差异,从而引起了各国的广泛注意。几年来,有关这方面的研究报告已有几十篇之多,并已出版了专著。     

核磁共振技术在森林凋落物分解研究中的应用

凋落物分解是连接森林生态系统地上和地下部分的关键过程,影响着森林生态系统的养分循环,对全球碳(C)循环和森林生态系统生产力具有重要的调控作用。特别是,在当前全球气候变化的背景下,研究森林凋落物不同有机C组分的分解动态对于了解土壤有机质的形成、稳定性及其对气候变化的反馈效应至关重要。核磁共振技术(NMR)具有无损和非侵入等特点,是研究有机化合物结构的有效手段,已在凋落物分解研究中得到广泛应用。本文概述了NMR技术应用于森林凋落物分解过程的最新研究进展,包括森林凋落物有机C基团的NMR特征,凋落物分解过程中不同C基团的变化特征及其机理、凋落物分解过程的NMR评价指标等,并分析了NMR技术在凋落物分解研究中存在的问题和未来研究方向。NMR技术将有助于了解凋落物有机C基团的分解特征及其对土壤有机质形成和稳定的影响机理。     

KUJIRA,a package of integrated modules for systematic and interactive analysis of NMR data directed to high-throughput NMR structure studies

The recent expansion of structural genomics has increased the demands for quick and accurate protein structure determination by NMR spectroscopy. The conventional strategy without an automated protocol can no longer satisfy the needs of high-throughput application to a large number of proteins, with each data set including many NMR spectra, chemical shifts, NOE assignments, and calculated structures. We have developed the new software KUJIRA, a package of integrated modules for the systematic and interactive analysis of NMR data, which is designed to reduce the tediousness of organizing and manipulating a large number of NMR data sets. In combination with CYANA, the program for automated NOE assignment and structure determination, we have established a robust and highly optimized strategy for comprehensive protein structure analysis. An application of KUJIRA in accordance with our new strategy was carried out by a non-expert in NMR structure analysis, demonstrating that the accurate assignment of the chemical shifts and a high-quality structure of a small protein can be completed in a few weeks. The high completeness of the chemical shift assignment and the NOE assignment achieved by the systematic analysis using KUJIRA and CYANA led, in practice, to increased reliability of the determined structure.     

Flow NMR applications in combinatorial chemistry

Flow NMR techniques are now well accepted and widely used in many areas of drug discovery. Although natural-product-, rational-drug-design-, and NMR-screening-programs have begun to use flow NMR more routinely, flow NMR has not yet gained widespread acceptance in combinatorial chemistry, even though it has been shown to be a potentially useful tool. Recent developments in DI-NMR, FIA-NMR, and LC-NMR will help flow NMR eventually gain a wider acceptance within combinatorial chemistry. These developments include LC-NMR-MS instrumentation, flow probe improvements, new pulse sequences, improved automation of NMR data analysis, and the application of flow NMR to related fields in drug discovery.     

High-field NMR as a technique for the determination of polysaccharide structures

NMR spectroscopy has played a developing role in the study of polysaccharide structures for over 30 years. Many new bacterial polysaccharide repeat unit structures have recently been published as a result of the application of modern NMR techniques. NMR can also be used to elucidate the structures of both regular and heterogeneous polysaccharides from fungal and plant sources, as well as complex glycosaminoglycans of animal origin. In addition to covalent structure, conformation and dynamics of polysaccharides are susceptible to NMR analysis, both in solution and in the solid state. Improvements in NMR technology with potential applications to polysaccharide studies hold promise for the future.     

Mollib: a molecular and NMR data analysis software

Mollib is a software framework for the analysis of molecular structures, properties and data with an emphasis on data collected by NMR. It uses an open source model and a plugin framework to promote community-driven development of new and enhanced features. Mollib includes tools for the automatic retrieval and caching of protein databank (PDB) structures, the hydrogenation of biomolecules, the analysis of backbone dihedral angles and hydrogen bonds, and the fitting of residual dipolar coupling (RDC) and residual anisotropic chemical shift (RACS) data. In this article, we release version 1.0 of mollib and demonstrate its application to common molecular and NMR data analyses.     

A nuclear magnetic resonance imaging technique designed for studies of water in plant leaves.

A new imaging technique is described which uses nuclear magnetic resonance (NMR) to create a water profile of plant leaves. The water profile shows the average distribution of water as a function of depth into a leaf along a line perpendicular to the leaf surface; it can be used to measure the thickness of cell layers and the quantity of water in each layer. Two-dimensional NMR methods were used to avoid chemical shift distortion which degrades the resolution in leaf images made by conventional NMR techniques; image resolution was improved further by deconvolution analysis. To illustrate its application, the technique was used to follow changes in the internal structure of developing leaves.     

COCO: a simple tool to enrich the representation of conformational variability in NMR structures

NMR structures are typically deposited in databases such as the PDB in the form of an ensemble of structures. Generally, each of the models in such an ensemble satisfies the experimental data and is equally valid. No unique solution can be calculated because the experimental NMR data is insufficient, in part because it reflects the conformational variability and dynamical behavior of the molecule in solution. Even for relatively rigid molecules, the limited number of structures that are typically deposited cannot completely encompass the structural diversity allowed by the observed NMR data, but they can be chosen to try and maximize its representation. We describe here the adaptation and application of techniques more commonly used to examine large ensembles from molecular dynamics simulations, to the analysis of NMR ensembles. The approach, which is based on principal component analysis, we call COCO ("Complementary Coordinates"). The COCO approach analyses the distribution of an NMR ensemble in conformational space, and generates a new ensemble that fills "gaps" in the distribution. The method is very rapid, and analysis of a 25-member ensemble and generation of a new 25 member ensemble typically takes 1-2 min on a conventional workstation. Applied to the 545 structures in the RECOORD database, we find that COCO generates new ensembles that are as structurally diverse-both from each other and from the original ensemble-as are the structures within the original ensemble. The COCO approach does not explicitly take into account the NMR restraint data, yet in tests on selected structures from the RECOORD database, the COCO ensembles are frequently good matches to this data, and certainly are structures that can be rapidly refined against the restraints to yield high-quality, novel solutions. COCO should therefore be a useful aid in NMR structure refinement and in other situations where a richer representation of conformational variability is desired-for example in docking studies. COCO is freely accessible via the website www.ccpb.ac.uk/COCO.     

Determination of full 13C isotopomer distributions for metabolic flux analysis using heteronuclear spin echo difference NMR spectroscopy

13C-isotopomer labeling experiments play an increasingly important role in the analysis of intracellular metabolic fluxes for genetic engineering purposes. 13C NMR spectroscopy is a key technique in the experimental determination of isotopomer distributions. However, only subsets of isotopomers can be quantitated using this technique due to redundancies in the scalar coupling patterns and due to invisibility of the 12C isotope in NMR. Therefore, we developed and describe in this paper a 1H NMR spectroscopy method that allows to determine the complete isotopomer distribution in metabolites having a backbone consisting of up to at least four carbons. The proposed pulse sequences employ up to three alternately applied frequency-selective inversion pulses in the 13C channel. In a first application study, the complete isotopomer distribution of aspartate isolated from [1-13C]ethanol-grown Ashbya gossypii was determined. A tentative model of the central metabolism of this organism was constructed and used for metabolic flux analysis. The aspartate isotopomer NMR data played a key role in the successful determination of the flux through the peroxisomal glyoxylate pathway. The new NMR method can be highly instrumental in generating the data upon which isotopomer labeling experiments for flux analysis, that are becoming increasingly important, are based.     

Probing alignment and phase behavior in intact wood cell walls using 2H NMR spectroscopy

This study presents the first application of (2)H NMR spectroscopy to quantify lignocellulose matrix orientation, and it demonstrates the ability to separately investigate oriented and unoriented amorphous domains in intact natural plant tissue. Matrix orientation is evaluated using NMR quadrupolar interactions in small deuterated probe molecules absorbed into bulk Liriodendron tulipifera sapwood. Ethylene glycol-d(4) deuterium spectra reveal two distinct amorphous domains, a highly oriented phase in the secondary wall S2 layer and an isotropic domain probably reflecting the compound middle lamella (CML). The oriented and isotropic signal areas exhibit thermally reversible changes, postulated to reflect probe redistribution between the S2 layer and the CML. Preliminary studies on a more powerful wood swelling agent, N,N-dimethylformamide-d(1), are also discussed. This (2)H NMR technique provides a new avenue for analysis and understanding of lignocellulose ultrastructure and promises to create new insights in correlating biomass processing with morphological change.     

生物大分子质谱电离技术的突破及核磁共振三维结构测定方法的建立——2002年诺贝尔化学奖简介

2002年诺贝尔化学奖授予了质谱和核磁共振领域的三位科学家以表彰他们对生物大分子鉴定及结构分析方法做出的贡献．其中两位科学家J．B．Fenn和K．Tanaka分别发展了生物大分子质谱分析的软解吸电离方法;另一科学家K．Wüthrich则将核磁共振技术成功地应用于生物大分子如蛋白质的溶液三维结构测定．他们的研究成果已使质谱和核磁共振技术成为生物大分子强有力的研究手段,极大地促进了生物大分子的研究进程,必将对整个生命科学研究产生深远的影响．