Slow 5F-Trp Dynamics in a Kinetically Trapped Thermodynamically Unstable Protein Probed by Quantitative ¹⁹ F CEST and Real Time Unfolding NMR Measurements

Using quantitative phase-cycled ¹⁹F CEST NMR measurements on a thermodynamically unstable, and kinetically trapped 5F-Trp labeled F126W mutant variant of the isolated C terminal domain (CTD) of the fold switching protein RfaH, which globally unfolds over days, we show that the ¹⁹F nucleus exchanges between a buried (major) and (sparsely populated) solvent exposed environment on the millisecond-second time scale in the kinetically trapped native state. The analysis of lengthy ¹⁹F CEST measurements to quantify the slow exchange kinetics is complicated by the very slow global unfolding of the protein but can be alleviated by the addition of the osmolyte TMAO (trimethylamine <i>N</i>-oxide) and a simple apodization of the CEST baseline. The distinction of the visible (minor) globally unfolded species from the invisible sparsely populated conformation in the native state ensemble with indistinguishable ¹⁹F chemical shifts is facilitated by a combination of real-time unfolding, ¹⁹F CEST and EXSY measurements, and line width analysis.