NMR scalar couplings across Watson–Crick base pair hydrogen bonds in DNA observed by transverse relaxation-optimized spectroscopy

Abstract

H]-transverse relaxation-optimized spectroscopy (TROSY) (3-5) of scalar couplings across the Watson-Crick base pairs in isotope-labeled DNA, which affords direct observation of the hydrogen bonds in these structures. Scalar couplings across hydrogen bonds have been previously reported for organicsynthetic compounds (6, 7), RNA fragments (8), and a metalloprotein (9, 10). The variability of such couplings observed so far indicates that they may become sensitive new parameters for detection of hydrogen bond formation and associated subtle conformational changes. Furthermore, in conjunction with quantum-chemical calculations, precise measurements of scalar couplings across hydrogen bonds can be expected to provide novel insights into the nature of hydrogen bonds in chemicals and in biological macromolecules. MATERIALS AND METHODSFully and partially 13 C, 15 N-doubly labeled DNA oligomers were synthesized on a DNA synthesizer (Applied Biosystems model 392-28) by the solid-phase phosphoroamidite method, by using isotope-labeled monomer units that had been synthesized according to a previously described strategy (11). Approximately 1 mol of oligomer was obtained from 5 mol of nucleoside bound to the resin. NMR samples of the DNA duplex at a concentration of Ϸ2 mM were prepared in 90% H 2 O͞10% D 2 O containing 50 mM potassium phosphate and 20 mM KCl at pH 6.0. NMR measurements were performed at 15°C on Bruker DRX500 and DRX750 spectrometers equipped with H bond length, the solid-state NMR value of 0.11 nm for G and T in a hydrated DNA duplex (19) was used. Relaxation of the imino proton due to dipole-dipole (DD) coupling with remote protons in the DNA duplex was represented as follows (2): in the Watson-Crick AAT pair by an adenosine amino proton at a distance of 0.24 nm and the adenosine C2 proton at 0.3 nm; in G'C by a guanosine amino proton at 0.22 nm and a cytosine amino proton at 0.25 nm. For both base pairs, two imino protons in sequentially stacked bases at 0.4 nm also were considered. Following the calculations outlined in refs. 3-5, the use of TROSY at a polarizing magnetic field B o ϭ 17.6 T is expected to yield 65% and 30% reductions of the 15 N and 1 H linewidth, respectively, for AAT base pairs and 55% and 20% reductions for G'C base pairs. If the contributions from dipolar interactions with remote protons are neglected, the calculations predict reductions of 85% and 75% for 15 N and 1

A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated –1 ribosomal frameshifting

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Nuclear Magnetic Resonance-Based Modeling and Refinement of Protein Three-Dimensional Structures and Their Complexes

“…Hydrogen bond restraints should be used with caution, although they can be very useful in the case of large proteins when not enough NOE data is available yet. Note that hydrogen bonds can now also directly be detected from cross-hydrogen bond scalar coupling measured from constant time HNCO spectra [25,26]. These can provide useful restraints for structure calculations [27].…”

Unravelling Glycobiology by NMR Spectroscopy

“…In order to overcome respectively these two obstacles, methods of in vitro or in vivo isotopic labeling with specific magnetically active nuclear spins (principally 13 C and 15 N) [2,3] and new pulse sequences to specifically reduce losses from transverse relaxation mechanisms [4][5][6] were developed along the years. These methods, established primarily to proteins and nucleic acids [4][5][6][7][8][9], are now being used to study carbohydrates and glycoconjugates as well [10,11].…”