Protein Dynamics with Sample-Shuttling Relaxometry

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A proper description of biological processes at the atomic level require a full characterization of both the structure and the dynamics of biomolecules. Nuclear magnetic resonance (NMR) is a method of choice to access both to the structure and the dynamics of proteins and nucleic acids. One of most powerful NMR probes of biomolecular dynamics is nuclear spin relaxation. Here, we show that nanosecond time scale motions can be revealed with an emerging technique: high-resolution relaxometry.

Conventional relaxation experiments are performed on high-field magnets that provide atomic resolution information on dynamics but rely on a limited sampling of motions in the frequency domain. In order to obtain more information in the frequency domain, A. G Redfield introduced a new NMR method called high-resolution relaxometry in 2003.

High-resolution relaxometry is a method where the polarization and the detection take place at high field while the relaxation occurs at low fields, in the stray field of a commercial magnet. A sample shuttle is used for the fast transfer between the high- and low-field positions. The stray field of such a magnet covers about three orders of magnitude of magnetic field, so that molecular motions can be directly sampled over an unprecedented range of frequencies.

Here, we report the design and performance of a new shuttle device installed on a 600 MHz spectrometer. We applied our methods to the protein ubiquitin for the measurement of longitudinal nitrogen-15 relaxation rates over nearly two orders of magnitude from 14.1T to 0.5T.

In order to analyze our data at high field and correct for the effects of relaxation during sample motions, we developed an iterative protocol called Iterative Correction and Analysis of Relaxation Under Shuttling  (ICARUS).

Our system allowed us to better quantify nanosecond motions in key regions of ubiquitin, such as the β1- β2 turn. This study opens the way to a quantitative characterization of nanosecond motions in proteins with high-resolution relaxometry.