Single-step determination of protein substructures using dipolar couplings: aid to structural genomics.
Single-step determination of protein substructures using dipolar couplings: aid to structural genomics.
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使用偶极耦合一步测定蛋白质子结构：有助于结构基因组学。
DOI：
10.1021/ja016496h
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发表时间：
2001
影响因子：
15
通讯作者：
Bax,A
中科院分区：
文献类型：
--
作者：
Zweckstetter,M;Bax,A
随着人类和各种其他基因组测序的完成，最近获得的大量基因组数据产生了快速有效确定相应蛋白质三维结构的需求。到目前为止，这个所谓的结构基因组学项目的大部分工作都集中在 X 射线晶体学上，但 NMR 也显示出巨大的潜力。 2, 3 传统上，NMR 结构测定由共振分配阶段组成，该阶段通常依赖于对大量三共振 J 连接数据的分析，随后是结构测定阶段，该阶段依赖于 NOE 谱的解释以及标量和偶极耦合的测量。 3, 4 在这里，我们描述并演示了一种更加综合的方法，其中分配和结构数据来自同一实验。这为大大加速 NMR 结构测定过程提供了机会。此处演示的方法依赖于 3D (HA) CANH 三重共振实验，5 将酰胺 1H 和 15N 化学位移与残基内和前面的 13CR 核的化学位移相关联。由于 13CR 化学位移通常不够独特，因此单独的谱不能用于确定完整的顺序分配。因此，经常使用将酰胺与残基内和前面的残基 13C' 或 13C 共振相关联的单独实验来完成分配过程。在这里，我们证明，如果以 1HR 耦合模式记录 (HA) CANH 光谱，则 JCRHR 分裂的大小和 13CR 化学位移各向异性 (CSA) 和 13CR-1HR 偶极耦合之间的弛豫干扰所导致的双峰强度6的不对称性都可以用来解决由于 13CR 化学位移的非唯一性引起的模糊性。当这样的实验在各向同性和液晶介质中进行时，所得的光谱不仅产生主链 1HN、15N 和 13CR 共振的完整分配，而且还包含 13CR-1HR 偶极耦合、13CR CSA 和随机螺旋 13CR 各向同性化学位移偏差形式的重要结构信息。总之，这提供了足够的信息来获得中小型蛋白质（例如泛素和钙调蛋白）的完整主链 1HN、15N 和 13CR 分配，并确定此类蛋白质片段的 3D 结构。为了避免 HR 耦合 (HA) CANH 光谱相对于常规 (HA) CANH 光谱的拥挤程度增加，两个 13CR-{1HR} 双峰组分通过计算总和和差以通常方式 7 分成两个单独的光谱
The wealth of genomic data that has recently become available with completion of the sequencing of both the human and a variety of other genomes1 has created a need for rapid and efficient determination of three-dimensional structures of the corresponding proteins. So far, most effort in this so-called structural genomics project has focused on X-ray crystallography, but NMR also shows considerable potential. 2, 3 Conventionally, NMR structure determination consists of a resonance assignment phase, which usually relies on analysis of an extensive set of triple resonance J-connectivity data, followed by a structure determination phase that relies on interpretation of NOE spectra and measurement of scalar and dipolar couplings. 3, 4 Here, we describe and demonstrate a more integrated approach, where the assignment and structural data are derived from the same experiment. This offers the opportunity to greatly accelerate the NMR structure-determination process.The method demonstrated here relies on the 3D (HA) CANH triple resonance experiment, 5 which correlates amide 1H and 15N chemical shifts with those of the intraresidue and preceding 13CR nuclei. Because the 13CR chemical shift is usually insufficiently unique, such a spectrum alone cannot be used for determining complete sequential assignments. Therefore, separate experiments that correlate the amide with intraresidue and preceding residue 13C′ or 13C resonances are frequently used to complete the assignment process. Here, we demonstrate that if the (HA) CANH spectrum is recorded in the 1HR-coupled mode, both the size of the JCRHR splitting and the asymmetry in doublet intensity6 resulting from relaxation interference between 13CR chemical shift anisotropy (CSA) and 13CR-1HR dipolar coupling can be used to resolve the ambiguities caused by the non-uniqueness of the 13CR chemical shift. When such an experiment is conducted in both an isotropic and a liquid crystalline medium, the resulting spectra not only yield complete assignment for the backbone 1HN, 15N, and 13CR resonances, but also contain important structural information in the form of 13CR-1HR dipolar coupling, 13CR CSA, and deviations from random-coil 13CR isotropic chemical shifts. Together, this provides sufficient information to obtain complete backbone 1HN, 15N, and 13CR assignments of small and mediumsized proteins, such as ubiquitin and calmodulin, and to determine the 3D structure of fragments of such proteins. To avoid increased crowding in the HR-coupled (HA) CANH spectrum relative to the regular (HA) CANH spectrum, the two 13CR-{1HR} doublet components are separated into two separate spectra in the usual manner7 by calculating the sum and difference