Complete assignment of the imino protons of Escherichia coli valine transfer RNA: two-dimensional NMR studies in water

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Abstract
The imino proton spectrum of E. coli Val tRNA was studied by 2-dimensional nuclear Overhauser effect spectroscopy (NOESY) in H2O solution. The small nuclear Overhauser effects from the imino proton of an internal base pair to the imino protons of each nearest neighbor can be observed as off-diagonal cross-peaks. In this way most of the sequential NOE connectivity trains for all the helices in this molecule can be determined in a single experiment. AU resonances can be distinguished from GC resonances by the AU imino NOE to the aromatic adenine C2-H, thus leading to specific base-pair assignments. In general, the NOESY spectrum alone is not capable of assigning every imino proton resonance even in well-resolved tRNA spectra. Multiple proton peaks exhibit more than 2 cross-peaks, resulting in ambiguous connectivities, and coupling between protons with similar chemical shifts produces cross-peaks that are incompletely resolved from the diagnoal. The sequence of the particular tRNA determines the occurrence of the latter problem, which can often be solved by careful 1-dimensional experiments. The complete imino proton assignments of E. coli Val tRNA are presented.