Probing Submillisecond-to-Millisecond Time Scale Conformational Dynamics in High-Molecular-Weight Biomolecules via ¹⁵ N Clean Transverse-Relaxation-Optimized Chemical Exchange Saturation Transfer (TROSY-CEST)

Chemical exchange saturation transfer (CEST) NMR experiments are widely employed to probe submillisecond-to-millisecond time scale motions, which are critical for a broad range of biological functions. The ¹⁵N TROSY-CEST variant, which leverages ¹⁵N spin probes in backbone amide groups and transverse-relaxation-optimized spectroscopy (TROSY), is especially advantageous for investigating high-molecular-weight biomolecules. However, this experiment is often compromised by spurious dips at anti-TROSY positions, which obscure true minor dips that report on conformational exchange processes. Here, we present an optimized protocol, called clean TROSY-CEST, for suppressing anti-TROSY artifacts in ¹⁵N TROSY-CEST experiments, which enhances the reliability of exchange detection compared with that of other conventional ¹⁵N CEST methods. Notably, the overlap between major dip from ground state and minor dip from excited state is significantly reduced, thereby facilitating the identification of exchange processes involving small chemical shift differences. This optimized scheme enables more accurate characterization of biologically relevant dynamics in high-molecular-weight biomolecules.