A comparison of the ROESY and NOESY experiments for large molecules, with application to nucleic acids

Abstract

Rotating-frame experiments, in particular TOCSY or HOHAHA, have found increasing use in proton NMR of macromolecules, substantially extending the molecular weight range for which complete assignments are possible ( 1-4). The theory and application of HOHAHA to proteins and DNA have been comprehensively described (57), but rather less attention has been paid to the rotating-frame NOE experiment (variously known as CAMELSPIN and ROESY). Bothner-By and co-workers (8) have shown that the rotating-frame cross-relaxation rate constant is positive for all correlation times. This allows dipolar interactions to be detected for molecules of any size, even those of intermediate molecular weight where the NOE is near zero. A second advantage of the ROESY experiment is that for large macromolecules, the effects of spin diffusion are reported to be less marked than in the corresponding NOESY experiment ( 9,lO). In the ROESY experiment, transfer of magnetization along a chain of spins is accompanied by a change of sign, similar to the three-spin effect for longitudinal NOES in the extreme-narrowing limit. It should therefore be possible to distinguish between direct and indirect transfers of magnetization (4). Further, multiple-step transfers are rapidly attenuated, so that the ROESY spectra can be simpler than the corresponding NOESY spectra. These properties are of particular importance in studies of nucleic acids, where tight dipolar coupling among the sugar protons and between the sugar protons and the base protons ensures that spin diffusion is severe at short mixing times when w7 $ 1 ( I I-1 7). The rapid onset of spin diffusion in nucleic acids and large proteins and the resulting lack of specificity of the NOE may reduce the reliability of assignments that rely on NOESY spectra. For the same reason, the interpretation of NOESY cross-peak intensities in terms of internuclear distances as required for structure determination by distance geometry and restrained molecular dynamics methods ( 18, 19) becomes very uncertain. Although these difficulties can be overcome, for example, by recording a series of NOESY spectra at several different mixing times and analyzing the time dependence in multispin calculations, the different time dependence of the ROESY cross-peak intensities suggests that this experiment may be a useful adjunct to the NOESY experiment for large molecules. Recently, Farmer et al. (20) have compared NOE intensities expected for the NOESY and ROESY experiments for AX and AX3 spin systems in the absence and