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

氢键（h -键）对于维持蛋白质和核酸的三维构象至关重要，在配体分子的识别和酶促反应的调节中起着关键作用。许多这样的配体含有磷酸基团；例如核苷酸、磷脂、辅酶A （CoA）、NAD及其衍生物、DNA和RNA。在配体磷酸盐和蛋白酰胺基团之间形成的分子间氢键通常对它们的相互作用以及酶促过程至关重要。然而，通过核磁共振鉴定这些氢键是很困难的，这些键通常是从预定结构中氢键供体和受体的空间接近度推断出来的，或者是从间接证据推断出来的，比如与溶剂的氢交换率降低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在N- h…O^—P氢键上的偶联。N-H…O=C‘氢键的^<3h>J_<NP>和^<2h>J_<HP>的大小比相应的*键和2键J在N-H…O=C’氢键上的连接大几倍。这些差异可能是由于受体基团的化学性质不同，因为两种类型的氢键长度d （NO）是相似的。在检测到^<3h>J_<NP>的存在后，我们使用了一个新的脉冲方案，^<3h>J_<NP> HNPO，来观察氢键供体和受体基团的^1H_N， ^<15>N和^<31>P之间的相关性。由于^<3h>J_<NP>的大尺寸，HNPO实验的灵敏度显着高，并且由于^<31>P的高自然丰度，只需要^<15>N标记。这些因素使得HNPO实验对于研究蛋白质与含磷酸盐配体（包括DNA和RNA）的相互作用以及它们复合物的结构确定特别有价值，因为它可以明确地识别氢键供核和接受核。

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.