Mesodynamics in the SARS nucleocapsid measured by NMR field cycling

Clarkson, Michael W.; Lei, Ming; Eisenmesser, Elan Z.; Labeikovsky, Wladimir; Redfield, Alfred; Kern, Dorothee

The dynamic nature of proteins is one of the key elements of their function. Although this principle is widely accepted, the detailed characterization of protein motions at atomic resolution is much more challenging than determining the average static structure. High field NMR is a particularly powerful method for studying protein dynamics at atomic resolution over a wide array of time scales and has therefore extensively been used in the last two decades. The major emphasis on the development of ultra-high-field magnets for an increase in sensitivity and resolution has allowed investigations on ever-larger biomolecular systems. Ironically, this high-field regime is not as sensitive to mesodynamics as relaxation data at lower fields. Low-field R 1 relaxation experiments covering 1 H Larmor frequencies up to several tens of MHz have been performed using fieldcycling relaxometers (Kimmich 1996; Luchinat and Parigi 2007) . Due to the low resolution, these experiments have usually probed the relaxation of solvent protons as reporters, and are therefore limited. Here we report R 1 relaxation data of a protein at low field, but with atomic resolution, using a field-cycling apparatus in a commercial 500 MHz magnet (Redfield 2003) . By physically moving the sample in the magnet bore the relaxation behavior can be investigated at various low fields; however, the return of the sample to its initial position allows these measurements with the resolution and sensitivity of the high field. This approach has been successfully applied to studies of lipids and DNA, using one-dimensional 31 P and 13 C spectra (Roberts et al. 2004; Sivanandam et al. 2009 ). Here we use two-dimensional 1 H 15 N heteronuclear experiments spanning from 17.3 to 91.2 MHz ( 15 N Larmor frequency will be used throughout, corresponding to 170-900 MHz ''spectrometers'') to investigate the dynamics of the N-terminal domain of the SARS (Severe Acute Respiratory Syndrome) nucleocapsid protein (SARSN).