Differential deuterium isotope shifts and one-bond 1H-13C scalar couplings in the conformational analysis of protein glycine residues.

The one-bond deuterium isotope shift effect for glycine C alpha resonances exhibits a conformational dependence comparable to that of the corresponding 1JHC scalar coupling in both magnitude (approximately 11 Hz at 14.1 T) and dihedral angle dependence. The similarity in the conformational dependence of the 1JHC and deuterium isotope shift values suggests a common physical basis. Given the known distribution of (phi, psi) main-chain dihedral angles for glycine residues, the deuterium isotope shifts and the 1JHC scalar couplings can determine conformations in the left- and right-handed helical-to-bridge regions of the (phi, psi) plane to an accuracy of approximately 13 degrees. In the absence of stereochemical assignments, the differential deuterium isotope shifts and the 1JHC scalar couplings can be combined with limited independent structural information (e.g., the sign of phi) to determine the chirality of the deuterium substitution.

Resolution and sensitivity enhancement of heteronuclear correlation for methylene resonances via 2H enrichment and decoupling.

Monodeuterated methylene positions exhibit substantially superior spectral characteristics in 1H-13C correlation experiments as compared to diprotio signals. A combination of 2H decoupling and multiplet editing of HMQC and HSQC experiments provides resolution enhancement for both stereoselective and random fractionally deuterated samples. For HMQC experiments with [2-2HR,2-13C]glycine-labeled E. coli thioredoxin (11.7 kDa), 3-fold increases in both 1H and 13C resolution result in a complementary 9-fold enhancement in sensitivity. Owing to a smaller improvement in 13C resolution, the corresponding enhancements for the HSQC experiment are 2-fold less.

Conformational properties of DNA hairpins with TTT and TTTT loops.

The DNA hairpins d[CGATCG-Tn-CGATCG] (n = 3, 4) have been studied by NMR in order to gain information on hairpin conformation and flexibility. Resonance assignments were made using a combination of DQF-COSY, DQF-COSY[31P], NOESY, and 1H-31P-COSY. These data also provide approximate coupling constant information which points out exceptionally flexible regions of the phosphate backbone. The data for both hairpins reveal substantial flexibility within the loop segments. For n = 4, NOESY data alone are insufficient to distinguish between two loop-folding motifs, although coupling constant data favor a conformation in which Tb is folded toward the minor groove and is highly exposed to solvent. This is in agreement with chemical shift data and susceptibility to modification by KMnO4. The phosphate backbone between Tc and Td is exceptionally flexible, undergoing a facile exchange between (beta t,gamma+) and (beta+,gamma t) conformers. A similar flexible phosphate is observed between Tc and C7 when n = 3. Differences in stem conformation and dynamics in both hairpins are restricted to the two base pairs adjacent to the stem-loop junction. The C7pG8 stem phosphate appears to flip easily between (zeta-,alpha-) and (zeta-,alpha t) conformers when n = 4 but not when n = 3. Hairpin loop size thus affects the conformational flexibility of the adjacent stem segment.

A novel perlactone from the Caribbean sponge Plakortis angulospiculatus.

The Caribbean sponge Plakortis angulospiculatus contains a perlactone, plakinidone, that was identified by interpretation of spectral data. Plakinidone is the first natural product that incorporates a six-membered perlactone ring.

Chemical synthesis of oligodeoxynucleotide dumbbells.

The chemical synthesis of DNA dumbbells is investigated by using two sequences, cyclo-d(GCG-T4-CGCCGC-T4-GCG) and cyclo-d(TTCC-T4-GGAATTCC-T4-GGAA). This method readily and inexpensively yields multimicromole quantities of circular DNA, allowing detailed structural and physical studies to be carried out. Linear oligomers of sequence d(GCG-T4-CGCCGC-T4-GCG) having either 3'- or 5'-phosphates were cyclized in 40% and 67% isolated yield, respectively, by using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide. Formation of the circular product is confirmed by a 28 degrees C increase in the optical melting temperature, anomalously rapid electrophoretic migration, sequential nuclear Overhauser enhancements between protons of G1 and G20, and observed nuclear coupling between the ligated phosphorus and protons of both G1 and G20. cyclo-d(TTCC-T4-GGAATTCC-T4-GGCC) was synthesized from the corresponding linear 3'-phosphate in 80% yield by the same procedure. Chemical ligation is most effective for 3'-phosphorylated nick sites flanked by two purine bases.