ROESY Spectrum (Molecular Biology)

A conventional nuclear Overhauser effect (NOE) experiment in nuclear magnetic resonance (NMR) spectroscopy involves detection of changes in the intensities of NMR signals associated with a particular spin of the molecule being examined, when other spins are perturbed in some way. The changes in signal intensity are the result of altered populations of nuclear spin energy levels associated with both sets of spins and reflect their physical proximity. There may be magnetic interactions between spins while they are in coherent states and these may lead to changes of signal intensity. This second kind of Overhauser effect is detected in a "rotating frame" NOE (or ROE) experiment. The ROE may be determined in a one-dimensional format or be incorporated into multidimensional experiments. A two-dimensional (2D) ROE experiment is often referred to as ROESY (rotating frame Overhauser effect spectroscopy). In such a 2D experiment, the appearance of a cross peak at the chemical shift coordinates (A, B) requires that there be a ROE between the spins characterized by these chemical shifts (sa, sb). The power of experiments that produce ROEs or NOEs is that changes in signal strength are strongly dependent on the distance between interacting spins. Thus, the observation of a ROE or NOE provides a constraint that can be used to define the tertiary structure of the macromolecule being studied.

For the standard NOE experiment wherein the detected signal intensity changes arise from alterations of spin energy level populations, the effect is given by


where fI[S} indicates the NOE on the signal from spin I when there is a perturbation of level populations associated with spin S: I 0 is the normal intensity of the signal for spin I observed from a sample at thermal equilibrium before the analyzing RF pulse, and I p is the intensity of the same signal when there has been a perturbation of spins S before the analyzing pulse. The value offj[S] depends on the gyromagnetic ratios of spins I and S, how these spins move in the sample, the strength of the magnetic field used for the NMR experiment, and the details of how the energy level populations associated with spin S are perturbed during the course of an experiment. Values offI[S} ranging from 0.5 to -1.0 are possible when both I and S are protons. It should be noted that, given the definition offI[S}, some set of experimental conditions may exist for whichfI[S} becomes zero.

Under these conditions, there is little or no change in signal intensity, even though spins I and S might be very close to each other. These conditions are typically experienced when the mass of the molecule under study is in the range of 500-2000 D.

In contrast, the ROE on signal intensities is always positive for near-neighbor nucleus-nucleus interactions and thus remains detectable under all experimental conditions (1-3). This very significant advantage is countered, however, by the need to correct the experimental data for various artifacts and by the possibility that coherence transfers may produce effects that are unrelated to the ROE. Analysis of ROE data in conjunction with conventional NOE data may provide important insights into existing chemical exchange processes (4). Although most ROESY experiments with biological macromolecules involve interactions between hydrogen 1H atoms, useful heteronuclear ROESY experiments also are feasible (5). The elements of the ROESY experiment can be built into experiments that produce three-dimensional or higher NMR spectra. (See also Nuclear Overhauser Effect (NOE), NOESY Spectrum.)

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