用二维NMR交换谱定量地研究氘代DMSO和氘代氯仿中小肽活泼质子的交换动力学

用核磁共振二维交换谱方法研究了类肽化合物Cbz-Ser-Tyr-OC₆H₁₃中活泼质子的交换动力学,结果表明,在DMSO-d₆和CDCl₃溶液中,四种活泼质子与溶剂中残留水的交换快慢顺序为:Ser-OH > Tyr-OH > Ser-NH > Tyr-NH.在此基础上,对氨基酸中氨基所处的局部环境进行了讨论.     

白酒中氢键缔合作用的研究——乙醇-水缔合体中质子间的交换作用

本文报导用核磁共振(NMR)技术研究醇-水混合体系中发生缔合作用的质子间的交换作用过程。质子间的交换速度与醇-水混合的比例有关,在中性条件下,60%乙醇含量的水溶液,质子间交换速度最大。这种交换速度随着贮存期的延长而减缓,使得缔合体的平均寿命增长。质子间的交换速度对微量酸的存在非常敏感,酸可促使缔合平衡的更快建立,但要控制过量酸的存在对缔合体稳定性的影响。     

天然有机化合物NOESY谱的全弛豫矩阵分析

本文用定量处理2D NOE实验数据最精确的方法-全弛豫矩阵分析法定量分析了天然有机化合物显脉香茶菜素的相敏NOESY谱,计算出显脉香茶菜素分子中各质子间的交叉弛豫速率,根据1/r_tf⁶∝σ_tf计算出相应的质子间距离,结果表明:用全弛豫矩阵分析法计算出的质子间距与分子力学计算以及用单晶X-射线衍射法测得的质子间距离基本一致,为用核磁共振方法确定分子在溶液中的三维空间结构提供了可靠的结构参数。     

多肽和蛋白质NMR预饱和实验中饱和转移效应的研究

通过改变预饱和照射时间研究了一些多肽和蛋白质分子在水溶液中的饱和转移效应.定量分析了在照射60s之后大分子中绝大多数酰胺质子仍不受影响的原因.结果表明:具有稳定的三维溶液结构的蛋白质(如溶菌酶)中,活泼氢的信号基本上不受饱和转移影响;对于溶液结构比较稳定的多肽和蛋白质(如胰岛素),只有少部分酰胺质子信号强度受到影响;小分子六肽因为溶液中不存在稳定的构象,饱和转移效应十分显著.因此对于溶液中多肽与蛋白质构象的NMR研究,预饱和仍不失为一种简便而有效的溶剂峰压制技术.     

表面增强拉曼光谱对于蛋白质二级结构的酰胺III谱带表征

本文利用斜角沉积法制备银纳米棒阵列基底用于蛋白质二级结构的检测,结合酰胺Ⅲ谱带光谱分析,建立了一种基于表面增强拉曼光谱(SERS)蛋白质二级结构的表征方法.用这种方法获得了两种典型蛋白质(溶菌酶和细胞色素C)的SERS信号.通过分析蛋白质骨架酰胺Ⅲ的光谱,研究了浓度对蛋白质折叠状态的影响.结果表明在一定范围内随着浓度的增加,溶菌酶的α-螺旋结构和β-折叠结构成分增加,而细胞色素C的二级结构基本保持不变.     

表面增强拉曼光谱对于蛋白质二级结构的酰胺Ⅲ谱带表征（英文）

本文利用斜角沉积法制备银纳米棒阵列基底用于蛋白质二级结构的检测,结合酰胺Ⅲ谱带光谱分析,建立了一种基于表面增强拉曼光谱(SERS)蛋白质二级结构的表征方法.用这种方法获得了两种典型蛋白质(溶菌酶和细胞色素C)的SERS信号.通过分析蛋白质骨架酰胺Ⅲ的光谱,研究了浓度对蛋白质折叠状态的影响.结果表明在一定范围内随着浓度的增加,溶菌酶的α-螺旋结构和β-折叠结构成分增加,而细胞色素C的二级结构基本保持不变.     

有部分峰重叠时NOESY谱的全弛豫矩阵分析

采取用理论计算的NOESY峰强度代替NOESY谱中重叠的对角峰或交叉峰强度,用理论计算值和实验数据相结合的方法解决峰强度矩阵的完整化问题,用全弛豫矩阵分析法定量分析了谱中有部分峰重叠的天然有机化合物冬凌草乙素的相敏NOESY谱,计算出冬凌草乙素分子中各质子间的交叉弛豫速率,根据1/r_ij⁶∝σ_ij计算出相应的质子间距离,结果表明:用全弛豫矩阵分析法计算出的质子间距与分子力学计算得到的质子间距离完全一致。该方法能够用于谱中有部分峰重叠的天然有机化合物的NOESY谱的定量处理,可以为核磁共振方法确定分子在溶液中的三维空间结构提供可靠的结构参数     

基于CEST机制的pH成像方法、原理和应用

化学交换饱和转移（Chemical Exchange Saturation Transfer,CEST）技术作为一种新型的磁共振成像（Magnetic Resonance Imaging,MRI）技术.它的原理为溶质池中被激发饱和的质子与游离水中未被饱和的质子间的化学交换,能够引起水质子磁共振信号的下降,从而获得组织内生物分子的相关信息.由于质子间的交换速率k_ex与组织微环境的pH值之间存在直接联系,因而可以通过溶质质子的CEST信号对活体组织进行pH成像.目前用于pH成像的溶质分子既包括内源性游离的蛋白质、多肽分子,还包括一系列的外源性小分子和金属螯合物.通过不同类型的比率法、内源性胺和酰胺浓度-独立检测（Amine and Amide Concentration-independent Detection,AACID）等成像方法,能够获得肾脏、中风脑组织以及肿瘤组织的pH图谱.本文详细总结了2000年以来利用CEST技术进行pH成像方面的研究进展,包括对比剂、成像方法和相关应用,展望了活体组织pH成像的发展趋势和应用前景.     

Gd (DTPA-BIN)的热力学性质及与牛血清白蛋白作用的NMR研究

报道了一种新合成的DTPA酰胺类衍生物DTPA-BIN的质子解离过程,其质子化常数总和与稳定性大于DTPA-BMA并采用核磁共振弛豫分析法研究了Gd(DTPA、BIN)在水溶液和牛血清白蛋白溶液中的诱导弛豫增强性质.Gd(DTPA-BIN)配合物在水溶液中弛豫效率为3.28mmol^-1L·s^-1。牛血清白蛋白分子可以与Gd(DTP-BIN)配合物非共价地结合,这种非共价结合体的旋转相关时间明显长于自由配合物,使Gd(DTPA-BIN)在蛋白质溶液中弛豫效率增高.本文结果表明弛豫增强方法可以用来研究顺磁性金属配合物与蛋白质之间的相互作用情况.     

用二维NMR变换谱定量地研究氘代DMSO和氘代氯仿中小肽活泼质子的交换动力

用核磁共振二维交换谱方法研究了类肽化合物Ｃｂｚ－Ｔｙｒ－ＯＣ６Ｈ１３中活泼质子的交换动力学,结果表明,在ＤＭＳＯ－ｄ６和ＣＤＣｌ３溶液中,四种活泼质子与溶剂中残留水的交换快慢顺序为：Ｓｅｒ－ＯＨ〉Ｔｙｒ－ＯＨ〉Ｓｅｒ－ＮＨ〉Ｔｙｒ－ＮＨ,在此基础上,对氨基酸中氨基所处的局部环境进行了讨论。     

Residue-specific NH exchange rates studied by NMR diffusion experiments

We present a novel approach to the investigation of rapid (>2s(-1)) NH exchange rates in proteins, based on residue-specific diffusion measurements. (1)H, (15)N-DOSY-HSQC spectra are recorded in order to observe resolved amide proton signals for most residues of the protein. Human ubiquitin was used to demonstrate the proposed method. Exchange rates are derived directly from the decay data of the diffusion experiment by applying a model deduced from the assumption of a two-site exchange with water and the "pure" diffusion coefficients of water and protein. The "pure" diffusion coefficient of the protein is determined in an experiment with selective excitation of the amide protons in order to suppress the influence of magnetization transfer from water to amide protons on the decay data. For rapidly exchanging residues a comparison of our results with the exchange rates obtained in a MEXICO experiment showed good agreement. Molecular dynamics (MD) and quantum mechanical calculations were performed to find molecular parameters correlating with the exchangeability of the NH protons. The RMS fluctuations of the amide protons, obtained from the MD simulations, together with the NH coupling constants provide a bilinear model which shows a good correlation with the experimental NH exchange rates.     

NMR-Based amide hydrogen-deuterium exchange measurements for complex membrane proteins: development and critical evaluation

A method for measuring site-specific amide hydrogen-deuterium exchange rates for membrane proteins in bilayers is reported and evaluated. This method represents an adaptation and extension of the approach of Dempsey and co-workers (Biophys. J. 70, 1777-1788 (1996)) and is based on reconstituting (15)N-labeled membrane proteins into phospholipid bilayers, followed by lyophilization and rehydration with D(2)O or H(2)O (control). Following incubation for a time t under hydrated conditions, samples are again lyophilized and then solubilized in an organic solvent system, where (1)H-(15)N HSQC spectra are recorded. Comparison of spectra from D(2)O-exposed samples to spectra from control samples yields the extent of the H-D exchange which occurred in the bilayers during time t. Measurements are site specific if specific (15)N labeling is used. The first part of this paper deals with the search for a suitable solvent system in which to solubilize complex membrane proteins in an amide "exchange-trapped" form for NMR quantitation of amide peak intensities. The second portion of the paper documents application of the overall procedure to measuring site-specific amide exchange rates in diacylglycerol kinase, a representative integral membrane protein. Both the potential usefulness and the significant limitations of the new method are documented.     

β-环糊精与几种菁染料包合物的动态结构及包合平衡的NMR研究

用核磁共振方法研究了β-CD与三种菁染料3,3′-二（3-磺丙基）噻菁三乙胺盐(d-yeA)、3,3′-二（3-磺丙基）-5-氯-噻菁三乙胺盐(dyeB)和3,3′-二（3-磺丙基） -5,5′-二苯基-9-乙基恶碳菁(dyeC)在溶液中的包合现象和包合物的动态结构,实验发现β-CD环糊精疏水腔中的H-3和H-5以及三种菁染料的芳环上质子的化学位移由于形成包合物而发生明显变化, 而且峰的位置随包合程度的不同而不同. 实验中发现包合物的荧光强度随β-CD浓度增加而减弱,相应计算表明β-CD与三种菁染料形成了1∶1的包合物.本文研究了三种包合物中主体部分OH-3和OH-6的化学位移随温度变化的情况,获得了包合物中氢键强弱的信息, 通过NMR化学位移滴定法计算得到了三种包合物在水溶液中的稳定常数, 在上述工作的基础上,本文提出了三种包合物的结构模型,描述了客体进入主体腔中的动态过程.     

Effective rotational correlation times of proteins from NMR relaxation interference

Knowledge of the effective rotational correlation times, tauc, for the modulation of anisotropic spin-spin interactions in macromolecules subject to Brownian motion in solution is of key interest for the practice of NMR spectroscopy in structural biology. The value of tauc enables an estimate of the NMR spin relaxation rates, and indicates possible aggregation of the macromolecular species. This paper reports a novel NMR pulse scheme, [15N,1H]-TRACT, which is based on transverse relaxation-optimized spectroscopy and permits to determine tauc for 15N-1H bonds without interference from dipole-dipole coupling of the amide proton with remote protons. [15N,1H]-TRACT is highly efficient since only a series of one-dimensional NMR spectra need to be recorded. Its use is suggested for a quick estimate of the rotational correlation time, to monitor sample quality and to determine optimal parameters for complex multidimensional NMR experiments. Practical applications are illustrated with the 110 kDa 7,8-dihydroneopterin aldolase from Staphylococcus aureus, the uniformly 15N-labeled Escherichia coli outer membrane protein X (OmpX) in 60 kDa mixed OmpX/DHPC micelles with approximately 90 molecules of unlabeled 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), and the 16 kDa pheromone-binding protein from Bombyx mori, which cover a wide range of correlation times.     

N—甲基异靛蓝在不同溶剂中的^1H—NMR研究

用选择照射方法研究了N-甲基异靛蓝(isoindigatin)在(CD_3)_2CO、CDCl_3、DMSO-d_6溶剂中及重水交换后的~1H NMR谱。得到了化学环境极为类似的两芳环质子在不同溶剂中的NMR谱线归属和偶合常数。     

含Cr (Ⅲ)溶液中配位水质子的1H NMR研究

本文用¹H NMR对水合Cr(Ⅲ)络离子中配位水和质子交换速率进行研究。在溶液pH为0~3.17范围测得质子的平均寿命约为10^-5S。在pH为0~0.76范围质子交换反应动力学为一、二级混合过程。pH为0.76~1.6范围表现为一级过程。在Cr(Ⅲ)-H₂O-Gly系统中,实验得出配位水质子的活动性减小。     

1H NMR Study of Hydrogen Bonding and Hydrogen to Deuterium Isotope Exchange in Ureido Sugars

Abstract

The acidic properties of ureido NH protons in two new derivatives of methyl 3,4,6-triacetyl-2-deoxy-β-D-glucopyranoside and i-butylamine (1) and L-leucine (2) were studied by ¹H NMR. Stronger influence of polar aprotic solvents, more effective interaction with nitroxyl radical and faster H/D exchange indicate that N1'-H proton which is proximate to the glucopyranose ring is more acidic than N3'-H one in both ureido sugars, this conclusion was supported by semi-empirical AM1 calculation. Hydrogen to deuterium exchange rates are 1.2 x 10^?2 to 5.5x 10^?3 M^?1 min^?l. Significantly slower exchange of N3'-H proton standing by Leu residue in 2 can be explained by the existence of internal H-bonds.