| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1999: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
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.
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