Beyond slow two-state protein conformational exchange using CEST: applications to three-state protein interconversion...

Area of Science:

• Biophysics
• Structural Biology
• Chemical Physics

Background:

• Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful tool for studying biomolecular dynamics.
• Analyzing complex conformational exchange beyond two-state systems, especially on the millisecond timescale, presents significant challenges for standard NMR methods.
• Chemical Exchange Saturation Transfer (CEST) NMR experiments, initially designed for slow exchange, show promise for intermediate-fast timescale dynamics.

Purpose of the Study:

• To investigate the utility of the amide 15N CEST experiment for characterizing protein three-state exchange on the millisecond timescale.
• To study the interconversion dynamics between the folded (F) state and two minor folding intermediates (I1 and I2) of the FF domain from human HYPA/FBP11.
• To demonstrate that CEST NMR can resolve complex exchange processes beyond the capabilities of traditional CPMG experiments.

Main Methods:

• Utilized 15N CEST NMR experiments to probe millisecond timescale conformational exchange in the WT FF domain.
• Collected CEST data at multiple temperatures to enable robust three-state model fitting.
• Analyzed CEST data by incorporating the relative sign of chemical shift differences between minor states.

Main Results:

• 15N CEST experiments clearly identified a three-state exchange process involving the folded state and two minor states, unlike 15N CPMG experiments.
• A robust three-state model was established by analyzing multi-temperature CEST data, revealing interconversion rates for F, I1, and I2 states.
• The minor states (I1 and I2) were found to be sparsely populated (approx. 0.15% and 0.35% at 15°C) and interconvert on the millisecond timescale.
• Extraction of exchange parameters from CEST data did not require computationally intensive grid-searches.

Conclusions:

• 15N CEST NMR is highly effective for characterizing complex three-state protein conformational exchange on the millisecond timescale.
• The CEST experiment's utility extends to systems with interconversion rates in the thousands per second, surpassing limitations of slow exchange studies.
• This study validates CEST NMR as a valuable technique for detailed analysis of intricate biomolecular dynamics.