Dynamic 15N{1H} NOE measurements: a tool for studying protein dynamics

Vladlena Kharchenko,Andrzej Ejchart,Łukasz Jaremko,Michal Nowakowski,Mariusz Jaremko

doi:10.1007/s10858-020-00346-6

Abstract

Intramolecular motions in proteins are one of the important factors that determine their biological activity and interactions with molecules of biological importance. Magnetic relaxation of 15N amide nuclei allows one to monitor motions of protein backbone over a wide range of time scales. 15N{1H} nuclear Overhauser effect is essential for the identification of fast backbone motions in proteins. Therefore, exact measurements of NOE values and their accuracies are critical for determining the picosecond time scale of protein backbone. Measurement of dynamic NOE allows for the determination of NOE values and their probable errors defined by any sound criterion of nonlinear regression methods. The dynamic NOE measurements can be readily applied for non-deuterated or deuterated proteins in both HSQC and TROSY-type experiments. Comparison of the dynamic NOE method with commonly implied steady-state NOE is presented in measurements performed at three magnetic field strengths. It is also shown that improperly set NOE measurement cannot be restored with correction factors reported in the literature.

Highlights

Since its first use of magnetic relaxation measurements of 15N nuclei applied to the protein, the staphylococcal nuclease (Kay et al 1989), this method has become indispensable in the determination of molecular motions in biopolymers (Jarymowycz and Stone 2006; Kempf and Loria 2003; Palmer, III 2004; Reddy and Rayney 2010; Stetz et al 2019).Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.The canonical triad of relaxation parameters—longitudinal (R1) and transverse (R2) relaxation rates accompanied by the 15N{1H} nuclear Overhauser effect (NOE)—have been most often used in studies investigating the mobility of backbone in proteins
It has been shown that dynamic NOE measurement is an efficient and accurate method for NOE determination
It presents its usefulness in cases of NOE values that are close to zero

Summary

Introduction

Since its first use of magnetic relaxation measurements of 15N nuclei applied to the protein, the staphylococcal nuclease (Kay et al 1989), this method has become indispensable in the determination of molecular motions in biopolymers (Jarymowycz and Stone 2006; Kempf and Loria 2003; Palmer, III 2004; Reddy and Rayney 2010; Stetz et al 2019). The steady-state 15N{1H} NOEs (ssNOE) are normally determined as a ratio of cross-peak intensities in two experiments—with and without saturation of H N resonances. Such arrangement creates problems with computing statistically validated assessment of experimental errors. Unintentional irradiation of the water resonance suppresses HN and other exchangeable signals owing to the saturation transfer and many non-exchangeable 1H resonances via direct or indirect NOE with water (Grzesiek and Bax 1993) while interference of DD/CSA relaxation mechanisms of 15N amide nuclei disturbs the steady-state 15N polarization during 1H irradiation (Ferrage et al 2009). The dynamic NMR experiment (DNOE), a forgotten method of the NOE determination in proteins, was experimentally tested, and the results were compared with independently performed steady-state NOE measurements at several magnetic fields for widely studied, small, globular protein ubiquitin.

Results and discussion

Conclusions

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