Residue-specific NH exchange rates studied by NMR diffusion experiments.

Area of Science:

• Biophysics
• Protein Dynamics
• Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

• Investigating amide proton (NH) exchange rates in proteins is crucial for understanding protein dynamics and function.
• Rapid NH exchange (>2 s⁻¹) is challenging to measure accurately using conventional methods.
• Existing techniques may be limited in their ability to resolve residue-specific exchange rates.

Purpose of the Study:

• To present a novel approach for measuring rapid NH exchange rates in proteins.
• To demonstrate the utility of residue-specific diffusion measurements for this purpose.
• To correlate experimental findings with computational methods.

Main Methods:

• Utilized (1)H, (15)N-DOSY-HSQC spectra to observe amide proton signals.
• Applied a two-site exchange model with water, incorporating pure diffusion coefficients of water and protein.
• Determined protein's pure diffusion coefficient by suppressing water-to-amide proton magnetization transfer.
• Performed molecular dynamics (MD) and quantum mechanical calculations.

Main Results:

• Successfully measured rapid NH exchange rates in human ubiquitin.
• Demonstrated good agreement between experimental results and the MEXICO experiment for rapidly exchanging residues.
• Developed a bilinear model using RMS fluctuations from MD and NH coupling constants that correlates well with experimental exchange rates.

Conclusions:

• The proposed diffusion-based method is effective for investigating rapid protein NH exchange rates.
• The method provides residue-specific exchange rate information.
• Computational parameters like RMS fluctuations and coupling constants can predict NH exchange rates.