On the measurement of 15N–{1H} nuclear Overhauser effects

Abstract

Accurate quantification of the 15 N-{ 1 H} steady-state NOE is central to current methods for the elucidation of protein backbone dynamics on the fast, sub-nanosecond timescale. This experiment is highly susceptible to systematic errors arising from multiple sources. The nature of these errors and their effects on the determined NOE ratio is evaluated by a detailed analysis of the spin dynamics during the pair of experiments used to measure this ratio and possible improvements suggested. The experiment that includes 1 H irradiation, is analyzed in the framework of Average Liouvillian Theory and a modified saturation scheme that generates a stable steady state and eliminates the need to completely saturate 1 H nuclei, is presented. The largest source of error, however, in 1 H-dilute systems at ultra-high fields is found to be an overestimation of the steadystate NOE value as a consequence of the incomplete equilibration of the magnetization in the socalled "reference experiment". The use of very long relaxation delays is usually an effective, but time consuming, solution. Here, we introduce an alternative reference experiment, designed for larger, deuterated systems, that uses the fastest relaxing component of the longitudinal magnetization a closer approximation to the equilibrium state for shorter relaxation delays. The utility of the modified approach is illustrated through simulations on realistic spin-systems over a wide range of timescales and experimentally verified using a perdeuterated sample of human ubiquitin.

Rotational velocity rescaling of molecular dynamics trajectories for direct prediction of protein NMR relaxation

“…NOE measurements are notoriously sensitive to systematic bias. In particular, one may note a recent series of papers by Ferrage, Cowburn, Ghose and colleagues [46][47][48] analyzing the effects of the saturation scheme and water suppression techniques in quantifying protein 15 N NOE signals. They demonstrate that versions of the heteronuclear NOE experiment in common use can give rise to differences of 0.1.…”

“…Inaccuracies in the NOE measurements predominantly impact the estimated timescale of rapid internal motion which are generally recognized to have limited reliability. In contrast, errors on the order of 10% in the NOE generally lead to errors in the derived S 2 order parameters of only 1% [47]. An obvious potential benefit of direct MD-based prediction of NMR relaxation parameters would be to further stimulate the development and general implementation of more accurate heteronuclear NOE measurements.…”

Using NMR Spectroscopy to Investigate the Solution Behavior of Nerve Agents and Their Binding to Acetylcholinesterase

“…Because proton-13 C bond lengths are very similar from one bond to the next, the proton-13 C steady-state NOE values measured in NMR experiments are exclusively the result of molecular motions. In an analogous approach, the proton-15 N steady-state NOE is used to study the backbone dynamics of proteins (Ferrage et al, 2008). Figure 6.…”

On the measurement of 15N–{1H} nuclear Overhauser effects. 2. Effects of the saturation scheme and water signal suppression

“…Thus, a reassessment of the underlying spin-dynamics for these spin-relaxation experiments appears timely, to improve the accuracy by reducing deleterious effects and allowing for a proper interpretation of the measured relaxation rates in terms of protein dynamics. This is especially true for NOE's, which provide access to very high-frequency motional modes centered around the 1 H resonance frequency [13]. …”

“…These errors can be cancelled by two minor modifications to the experimental scheme: (1) applying 180° pulses to the 1 H nuclei to achieve effective saturation and (2) using delays between these 180° pulses that are matched to k/ J NH , where k is an integer [14]. In addition, we have also demonstrated, using Average Liouvillian Theory (ALT) [15; 16] that a symmetric saturation scheme obviates the need for a complete saturation of the 1 H magnetization [13]. This is especially advantageous for surface-exposed amide groups where 1 H N nuclei exchange with solvent 1 H nuclei.…”