A modified strategy for identification of 1H spin systems in proteins.
A modified strategy for identification of 1H spin systems in proteins.
复制标题
用于鉴定蛋白质中 1H 自旋系统的改进策略。
DOI：
10.1002/bip.360260615
复制
发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Wright,PE
中科院分区：
文献类型：
--
作者：
Chazin,WJ;Wright,PE
详细研究溶液中蛋白质的构象和动力学的'H-NMR光谱需要指定的光谱作为一个重要的第一步。获得这些任务的方法已开发的Wuthrich和同事的，并成功地应用于一些小的。在这份报告中，我们描述了一个修改的原始策略，这已被引入响应新的和复杂的实验技术的发展，并在研究较大的蛋白质系统中出现的特殊问题。本文所述的方法在我们的实验室中成功地应用于分子量为8000- 18，000 μ m的许多蛋白质和各种结构基序。获得顺序共振分配的基本方法涉及三个步骤的过程：1。使用二维标量相关实验（例如相关光谱（COSY））鉴定氨基酸自旋系统; 2.通过二维核奥弗豪泽效应光谱（NOESY）鉴定顺序相邻的残基; 3.将肽段分配到序列中的特定位置。此处所述的战略变化涉及第1级的分配，通常分两个阶段进行。首先，对于每种氨基酸自旋系统，从在H2O中获得的光谱中鉴定和分类侧链和C”质子共振。然后，有必要通过观察NH/C”H COSY在“H，O”中的交叉峰，将自旋系统的这一部分与不稳定的主链酰胺质子连接起来。我们在这里注意到，这把一个基本的重点放在确定连接到C”质子，从侧链和酰胺质子。在我们的新策略中，自旋系统识别的整体概念被修改，以便酰胺质子被用作分配的基础（Pro除外）。侧链和Ca质子通过在'H，O中直接建立与每个氨基酸自旋系统的酰胺质子共振的连接性来鉴定。识别前自旋系统，并且在必要时，使用来自H2O中的实验的补充数据扩展侧链分配并且细化自旋系统分类。在实验上，这种新策略的主要区别是对H，O记录的光谱的依赖性大大增加，并结合了最近开发的实验技术。这种改进策略的最初动力是众所周知的光谱重叠问题，该问题源于C”质子共振的相对有限的化学位移分散，特别是对于具有显著螺旋含量的蛋白质。该问题在图1中举例说明，图1显示了COSY谱的NH/C”H交叉峰区域的选定部分[图1（A）]和血清补体片段C3 a（77个氨基酸）的骨架酰胺和C”质子化学位移分布的比较[图1（B）]。在图1（A）中虚线描绘的小部分中，17个交叉峰分散在2 ppm的NH区域（w2）上，但仅分散在0.2 ppm的C”H区域（t0 1）范围内。在图1（B）中更一般地检查了该问题，该图比较了主链酰胺和C”质子的化学位移分布，并且清楚地表明，
Detailed studies of the conformation and dynamics of proteins in solution by'H-nmr spectroscopy requires assignment of the spectrum as an essential first step. The methodology for obtaining these assignments has been developed by Wuthrich and coworkers' and successfully applied to a number of small In this report we describe a modification to the original strategy, which has been introduced in response to the development of new and sophisticated experimental techniques, and to the special problems that arise in studying larger protein systems. The methodology described herein is being successfully applied in our laboratory to a number of proteins with molecular weights of 8000-18,000 d and a variety of structural motifs. The fundamental approach to obtaining sequential resonance assignments' involves a three-step process:1. identification of amino acid spin systems using two-dimensional scalar-correlated experiments (eg correlation spectroscopy (COSY)); 2. identification of sequentially adjacent residues by two-dimensional nuclear Overhauser effect spectroscopy (NOESY); 3. assignment of peptide segments to specific locations in the sequence. The change in strategy described here concerns assignments at the level of step 1, which are usually made in two separate stages. First, for each amino acid spin system, side-chain and C" proton resonances are identified and classified from spectra acquired in, H20. It is then necessary to connect this portion of the spin system to the labile backbone amide proton through observation of NH/C" H COSY cross-peaks in'H, O. We note here that this puts a fundamental emphasis on determining connectivities to the C" proton, from both the side-chain and amide protons. In our new strategy, the overall concept of spin system identification is modified so that the amide proton is used as a foundation for assignments (except for Pro). The side-chain and Ca protons are identified by establishing connectivities in'H, O directly to the amide proton resonance of each amino acid spin system. Pro spin systems are identified, and when necessary, side-chain assignments are extended and spin system classification refined Using complementary data from experiments in, H20. Experimentally, the principle difference in this new strategy is the considerably greater reliance on spectra recorded in'H, O and the incorporation of recently developed experimental techniques. The initial impetus for this modified strategy is the well-known problem of spectral overlap that arises from the relatively limited chemical-shift dispersion of C" proton resonances, particulivly for proteins with a significant helical content. The problem is exemplified in Fig. 1, which shows a selected part of the NH/C" H cross-peak region of a COSY spectrum [Fig. l (A)] and a comparison of the backbone amide and C" proton chemical-shift distributions [Fig. l (B)] for the serum complement fragment, C3a (77 amino acids). In the mall section delineated by dashed lines in Fig. 1 (A), 17 cross-peaks are dispersed over 2 ppm of the NH region (w2) but only over a range of 0.2 ppm of the C" H region (to1). The problem is examined more generally in Fig. l (B), which compares the chemical-shift distribution for the backbone amide and C" protons, and clearly
登录
查看更多内容
通过二维光谱法对大肠杆菌 lac 阻遏物 DNA 结合结构域 1-51 的 1H 核磁共振谱进行序列特异性共振分配。
DOI：
10.1111/j.1432-1033.1983.tb07827.x
发表时间：
1983
期刊：
European journal of biochemistry
影响因子：
--
作者：
E. Zuiderweg;R. Kaptein;K. Wüthrich
通讯作者：
K. Wüthrich
多肽和蛋白质 1H-NMR 谱中的连续个体共振分配
影响因子：
2.9
作者：
K. Wüthrich
通讯作者：
K. Wüthrich
兔肝金属硫蛋白-2 中序列特异性 1H-NMR 归属。
DOI：
10.1111/j.1432-1033.1986.tb09666.x
发表时间：
1986
期刊：
European journal of biochemistry
影响因子：
--
作者：
G. Wagner;D. Neuhaus;E. Wörgötter;M. Vašák;J. Kägi;K. Wüthrich
通讯作者：
K. Wüthrich
λ 的质子核磁共振研究。
DOI：
--
发表时间：
1985
期刊：
影响因子：
--
作者：
P. L. Weber;D. Wemmer;B. Reid
通讯作者：
B. Reid
通过中继磁化转移探索核自旋系统
DOI：
--
发表时间：
1982
期刊：
影响因子：
--
作者：
G. Eich;G. Bodenhausen;R. R. Ernst
通讯作者：
R. R. Ernst