Yasushi Oda

Binding of metal ions toE. coli RNase HI observed by1H−15N heteronuclear 2D NMR

SummaryThe divalent metal ion binding site and binding constant of ribonuclease HI fromEscherichia coli were investigated by observing chemical shift changes using1H−15N heteronuclear NMR. Chemical shift changes were monitored during the titration of the enzyme with salts of the divalent cations. The enzyme was uniformly labeled by15N, which facilitated the monitoring of the chemical shift change of each cross peak between the backbone amide proton and the amide15N. The chemical shifts of several amide groups were affected upon the addition of a divalent metal ion: Mg2+, Ca2+, or Ba2+. These amide groups resided close to the active site, consistent with the previous X-ray crystallographic studies. From the titration analysis, a single divalent ion binding site was observed with a weak binding constant (KD=2–4 mM for the current divalent ions).

1H NMR studies of deuterated ribonuclease HI selectively labeled with protonated amino acids.

SummaryTwo-dimensional (2D)1H NMR experiments using deuterium labeling have been carried out to investigate the solution structure of ribonuclease HI (RNase HI) fromEscherichia coli (E. coli), which consists of 155 amino acids. To simplify the1H NMR spectra, two fully deuterated enzymes bearing several prototed amino acids were prepared from an RNase HI overproducing strain ofE. coli grown in an almost fully deuterated medium. One enzyme was selectively labeled by protonated His, He. Val. and Leu. The other was labeled by only protonated His and Ile. The 2D1H NMR spectra of these deuterated R Nase H1 proteins, selectively labeled with protonated amino acids, were much more simple than those of the normally protonated enzyme. The simplified spectra allowed unambiguous assignments of the resonance peaks and connectivities in COSY and NOESY for the side-chain protons. The spin-lattice relaxation times of the side-chain protons of the buried His residue of the deuterated enzyme became remarkably longer than that of the protonated enzyme. In contrast, the relaxation times of the side-chain protons of exposed His residues remained essentially unchanged.

Design and synthesis of an α-helical peptide containing periodic proline residues

A thirty‐residue peptide (PERI COIL‐1) has been designed with a new type of α‐helical structure, which is capable of folding into an amphiphilic helix bending at 4 periodic prolines in the sequence. Two such helices should form a dimer by supercoiling about one another in an antiparallel direction in the design. With this arrangement, close packing between them is maintained through the hydrophobic interaction pattern called ‘leucine zipper’. PERI COIL‐1 has been obtained by solid‐phase peptide synthesis, and characterized by circular dichroic spectroscopy, sedimentation equilibrium experiments and NMR. The result of the analyses shows that it preferentially forms a helical tetramer in aqueous solution.

Synthesis and Properties of Dinucleoside Monophosphates Containing 2-Aminoadenine 8,2′-S- and Uracil 6,2′-O-Cyclonucleosides

Abstract Two sequence isomers of dinucleoside monophosphates containing 8,2′-anhydro-2,6-diamino-8-mercapto-9-β-D-arabinofuranosylpurine (2NH2As) and 6,-anhydro-6-hydroxy-1-s-D-arabinofuranosyluracil (Uo), 2NH2As pUo (1) and Uo p2NH2As (2) were synthesized by the phosphodiester method. Examination of the UV, CD and NMR spectra of these dimers led us to the conclusion that, whereas compound (1) did not take a stacked conformation, compound (2) took a well stacked conformation in which the bases were stacked along a left-handed screw axis. Both the dimers formed a complex with ethidium bromide.

NMR Studies of a DNA Containing 8-Methoxydeoxyguanosine

Abstract 1H NMR experiments have been undertaken to elucidate the structural effects of methoxy substitution at the C8 of a deoxyguanosine residue in a self-complementary dodecadeoxyribonucleotide, d(C-G-C-mo8G-A-A-T-T-C-G-C-G), duplex, which has an 8-methoxy-2′-deoxyguanosine (mo8dG) residue at the 4th position. NMR data indicate that the mo8dG residue takes an anti glycosidic conformation in a mo8dG(anti):dC(anti) base-pair structure in a B-form duplex. The thermal stability of the duplex is reduced, but the overall structure is much the same as that of the unmodified d(C-G-C-G-A-A-T-T-C-G-C-G) duplex.