OVERCOMING THE OVERLAP PROBLEM IN THE ASSIGNMENT OF H-1-NMR SPECTRA OF LARGER PROTEINS BY USE OF 3-DIMENSIONAL HETERONUCLEAR H-1-N-15 HARTMANN-HAHN MULTIPLE QUANTUM COHERENCE AND NUCLEAR OVERHAUSER MULTIPLE QUANTUM COHERENCE SPECTROSCOPY - APPLICATION TO INTERLEUKIN-1-BETA
OVERCOMING THE OVERLAP PROBLEM IN THE ASSIGNMENT OF H-1-NMR SPECTRA OF LARGER PROTEINS BY USE OF 3-DIMENSIONAL HETERONUCLEAR H-1-N-15 HARTMANN-HAHN MULTIPLE QUANTUM COHERENCE AND NUCLEAR OVERHAUSER MULTIPLE QUANTUM COHERENCE SPECTROSCOPY - APPLICATION TO INTERLEUKIN-1-BETA
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DOI：
10.1021/bi00441a004
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发表时间：
1989-07-25
期刊：
影响因子：
2.9
通讯作者：
CLORE, GM
中科院分区：
文献类型：
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作者：
MARION, D;DRISCOLL, PC;CLORE, GM
本文以分子量为17.4 kDa、153个残基的蛋白质(约95%)[15N]IL-1β为例，应用三维(3D)异核核磁共振波谱对较大蛋白质的~1H核磁共振谱进行了序列指认。白介素1β的二维(2D)600-MHz谱。由于广泛的交叉峰重叠和化学位移简并，太复杂而无法完成分析。我们发现，结合使用3D1H-15N Hartmann-Hahn-Multiple量子相干(HOHAHA-HMQC)和核Overhauser-Multiple量子相干(NOESY-HMQC)谱，为序列分配提供了必要的贯通键和贯通空间关联，为解决模糊问题提供了一种实用的通用方法，这严重限制了常规2D核磁共振谱的分析。这些3D谱中没有重叠的交叉峰，这使得可以毫不含糊地识别CαH(I)-NH(i+1)和NH(I)-NH(i+1)贯穿空间核Overhauser连接性，这是将特定的CαH-(I)-NH(I)贯穿键关联与其关联的贯穿空间顺序交叉峰连接所必需的。因此，有效地消除了1H核磁共振谱中酰胺NH化学位移简并的问题，分配过程仅涉及检查由直接键合的15N原子的化学位移编辑的一系列2D 1H-1H切片。残基之间的连接几乎可以在不知道所涉及的自旋系统类型的情况下被识别，尽管这种类型的信息显然是最终将连接的残基放置在初级序列中所必需的。获得自旋系统类型鉴定的策略包括传统的非活性质子谱分析、定点突变和选择的氨基酸的特定标记。预计3D异核光谱的内在简单性，即使对于150-200个残基的蛋白质，也将允许开发有效的计算机辅助或自动顺序分配方法。
The application of three-dimensional (3D) heteronuclear NMR spectroscopy to the sequential assignment of the 1H NMR spectra of larger proteins is presented, using uniformly labeled (.apprx.95%) [15N]interleukin 1.beta., a protein of 153 residues and molecular mass of 17.4 kDa, as an example. The two-dimensional (2D) 600-MHz spectra of interleukin 1.beta. are too complex for complete analysis, owing to extensive cross-peak overlap and chemical shift degeneracy. We show that the combined use of 3D 1H-15N Hartmann-Hahn-multiple quantum coherence (HOHAHA-HMQC) and nuclear Overhauser-multiple quantum coherence (NOESY-HMQC) spectroscopy, designed to provide the necessary through-bond and through-space correlations for sequential assignment, provides a practical general-purpose method for resolving ambiguities which severely limit the analysis of conventional 2D NMR spectra. The absence of overlapping cross-peaks in these 3D spectra allows the unambiguous identification of C.alpha.H(i)-NH(i+1) and NH(i)-NH(i+1) through-space nuclear Overhauser connectivities necessary for connecting a particular C.alpha.H-(i)-NH(i) through-bond correlation with its associated through-space sequential cross-peak. The problem of amide NH chemical shift degeneracy in the 1H NMR spectrum is therefore effectively removed, and the assignment procedure simply involves inspecting a series of 2D 1H-1H slices edited by the chemical shift of the directly bonded 15N atom. Connections between residues can be identified almost without any knowledge of the spin system types involved, though this type of information is clearly required for the eventual placement of the connected residues within the primary sequence. Strategies for obtaining identification of spin systems types include traditional analysis of the spectrum of nonlabile protons, site-directed mutagenesis, and specific labeling of selected amino acids. It is envisaged that the intrinsic simplicity of the 3D heteronuclear spectra, even for proteins of 150-200 residues, will permit the development of efficient computer-assisted or automated sequential assignment methods.