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NMR methods for determination of proteins : obtaining Information of long distances

用于测定蛋白质的 NMR 方法：获取长距离信息

基本信息

批准号：
11680662
负责人：
SHIRAKAWA Masao
金额：
$ 2.37万
依托单位：
NARA INSTITUTE OF SCIENCE AND TECHNOLOGY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Hydrogen bonds (H-bonds) are of central importance for maintenance of three-dimensional conformations of proteins and nucleic acids and play key roles in recognition of ligand molecules and in modulation of enzymatic reactions. Many such ligands contain phosphate groups ; examples are nucleotides, phospholipids, coenzyme A (CoA), NAD and its derivatives, DNA and RNA.Intermolecular H-bonds formed between the ligand phosphates and protein amide groups are usually crucial for their interactions, as well as for enzymatic processes. However, identifications of these H-bonds by means of NMR have been difficult, such bonds are usually inferred from the spatial proximity of the H-bond donor and acceptor in the pre-determined structure, and from indirect evidence, such as reduced hydrogen exchange rates with solvents and isotropic chemical shifts.We have detected intermolecular three-bond ^<31>P-^<15>N and two-bond ^<31>P-^1H J couplings across N-H…O^--P H-bonds. The magnitudes of ^<3h>J_<NP> and ^<2h>J_<HP> for the N-H…O^--P H-bonds are several-fold larger than corresponding *ond and 2-bond J connectivities across N-H…O=C' H-bonds of proteins. These differences are probably due to the different chemical nature of the acceptor groups, since the H-bond lengths, d (NO), are comparable for the two H-bond types. Having detected the presence of ^<3h>J_<NP>, we next used a new pulse scheme, ^<3h>J_<NP> HNPO, to observe correlations between ^1H_N, ^<15>N and ^<31>P of the H-bond donor and acceptor groups. Owing to the large size of ^<3h>J_<NP>, the sensitivity of the HNPO experiment is markedly high, and because of the high natural abundance of ^<31>P, only ^<15>N labeling is required. These factors should make this HNPO experiment particularly valuable for studying interactions of proteins with phosphate-containing ligands including DNA and RNA and for the structure determination of their complexes, as it can unambiguously identify both H-bond donating and accepting nuclei.

氢键（H-键）对于蛋白质和核酸的三维构象的维持至关重要，并且在配体分子的识别和酶促反应的调节中起关键作用。许多这样的配体含有磷酸基团;实例是核苷酸、磷脂、辅酶A（CoA）、NAD及其衍生物、DNA和RNA。配体磷酸和蛋白质酰胺基团之间形成的分子间氢键通常对于它们的相互作用以及对于酶促过程至关重要。然而，通过NMR来识别这些氢键是困难的，这些键通常是从氢键供体和受体在预定结构中的空间接近性来推断的，并且从间接证据来推断，例如与溶剂的降低的氢交换速率和各向同性化学位移。我们已经检测到分子间的三键^<31>P-^<15>N和两键^<31>P-^1H J耦合穿过N-H... O^-P H键。N-H<3h><NP><2h><HP>...O^--这些差异可能是由于受体基团的不同化学性质，因为两种氢键类型的氢键长度d（NO）相当。在检测到^ J_的存在之后<3h><NP>，我们接下来使用新的脉冲方案^<3h>J_<NP>HNPO来观察氢键给体和受体基团的^1H_N、^<15>N和^<31>P之间的相关性。由于^ J的尺寸很大<3h><NP>，HNPO实验的灵敏度非常高，并且由于^ P的天然丰度很高<31>，因此只需要^<15>N标记。这些因素应该使这个HNPO实验特别有价值的研究蛋白质与含磷酸盐的配体，包括DNA和RNA的相互作用，并为它们的复合物的结构测定，因为它可以明确地确定两个H-键捐赠和接受核。