Using the amide ¹⁵N CEST NMR experiment to study slow exchange between 'visible' protein states

Area of Science:

• Biochemistry
• Structural Biology
• Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

• Slow exchange between protein states is crucial for understanding protein function and dynamics.
• Traditional 2D ZZ exchange NMR experiments can suffer from spectral overlap, complicating analysis.
• Alternative methods are needed to accurately study protein dynamics and conformational changes.

Purpose of the Study:

• To evaluate the 15N CEST experiment as an alternative to 1H-15N ZZ exchange NMR for studying slow exchange between protein states.
• To demonstrate how 15N CEST can overcome spectral overlap issues inherent in ZZ exchange experiments.
• To establish a method for obtaining site-specific exchange parameters using 15N CEST.

Main Methods:

• Utilized the 15N CEST experiment to study exchange between protein states.
• Recorded two 15N CEST profiles per exchanging site in a two-state exchange system.
• Simultaneously analyzed 15N CEST profiles with initial magnetization or minor state population data.
• Applied the method to a T34A mutant of T4 lysozyme undergoing interconversion between native and minor states.

Main Results:

• The 15N CEST experiment effectively avoids spectral overlap encountered in ZZ exchange NMR.
• Site-specific exchange parameters were accurately determined by analyzing dual 15N CEST profiles.
• The method successfully characterized the exchange dynamics of T4 lysozyme mutant (18 kDa) between its native and minor states (21% population, ~5 s-1 exchange rate at 40°C).

Conclusions:

• 15N CEST is a powerful and advantageous alternative to ZZ exchange NMR for studying slow protein dynamics.
• This approach provides a robust method for characterizing exchange between 'visible' protein states with high precision.
• The study validates the utility of 15N CEST for detailed analysis of protein conformational exchange processes.