NMR relaxation analysis was used to characterize the internal dynamics of oxidized E. coli thioredoxin in both the picosecond-nanosecond and microsecond-millisecond frequency ranges for 413 H−C and H−N bond vectors. The 13C relaxation data was obtained utilizing protein samples possessing an alternating 13C−12C−13C... labeling pattern for most enriched sites. When combined with partial deuteration, this labeling pattern provides for isolated 1H−13C IS spin pairs exhibiting dynamically interpretable relaxation behavior. Side chains were found to exhibit a far broader range of dynamics than have been previously characterized for main chain resonances. The dynamics of structurally buried aromatic and leucine side chains are interpreted in terms of correlated main chain-side chain torsional oscillations. Structural regions exhibiting millisecond dynamics were found to correlate strongly with the presence of side chain-main chain or bifurcated main chain hydrogen bonds. Nuclei around the active site disulfide that exhibit mobility in the millisecond range correspond closely to the set of nuclei whose chemical shifts are altered upon reduction. The transient conformation is interpreted in terms of enhanced reactivity due to strain at the disulfide linkage.
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.
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.
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