Probing Submillisecond-to-Millisecond Time Scale Conformational Dynamics in High-Molecular-Weight Biomolecules via...

Area of Science:

• Biophysical Chemistry
• Structural Biology
• Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

• Chemical exchange saturation transfer (CEST) NMR probes molecular dynamics crucial for biological functions.
• ¹⁵N TROSY-CEST is valuable for studying high-molecular-weight biomolecules but suffers from anti-TROSY artifacts.
• These artifacts obscure important signals related to conformational exchange processes.

Purpose of the Study:

• To present an optimized protocol, clean TROSY-CEST, for suppressing anti-TROSY artifacts in ¹⁵N TROSY-CEST experiments.
• To enhance the reliability of detecting conformational exchange in biomolecules.
• To improve the characterization of biologically relevant dynamics in high-molecular-weight biomolecules.

Main Methods:

• Implementation of a novel clean TROSY-CEST protocol.
• Utilizing ¹⁵N spin probes in backbone amide groups.
• Leveraging transverse-relaxation-optimized spectroscopy (TROSY) for enhanced detection.

Main Results:

• Successfully suppressed anti-TROSY artifacts in ¹⁵N TROSY-CEST experiments.
• Significantly reduced overlap between major and minor dips, improving signal clarity.
• Enhanced the reliability of exchange detection compared to conventional ¹⁵N CEST methods.

Conclusions:

• The clean TROSY-CEST protocol offers a more reliable method for detecting exchange processes.
• Facilitates the identification of exchange processes with small chemical shift differences.
• Enables more accurate characterization of dynamics in large biomolecules using NMR.