A methyl ¹H double quantum CPMG experiment to study protein conformational exchange

Area of Science:

• Biophysics
• Structural Biology
• Protein Dynamics

Background:

• Protein conformational changes are essential for biological function.
• The Carr-Purcell-Meiboom-Gill (CPMG) experiment is used to study protein dynamics involving interconversion between states with lifetimes of 0.5–5 ms.
• Existing methyl 1H single and triple quantum relaxation dispersion experiments probe these dynamics.

Purpose of the Study:

• To develop a novel methyl 1H double quantum coherence experiment for studying protein dynamics.
• To enhance the accuracy of measuring protein exchange rates, particularly for fast processes.
• To complement existing CPMG-based relaxation dispersion techniques.

Main Methods:

• Development of a new NMR experiment utilizing methyl 1H double quantum coherences evolving during a CPMG relaxation element.
• Fitting of single, double, and triple quantum relaxation dispersion datasets.
• Application of the method to T4 lysozyme mutants and a fast-folding domain.

Main Results:

• The methyl double quantum experiment accurately determines exchange values, even for rates exceeding 10,000 s^-1.
• The method was successfully applied to T4 lysozyme mutants with varying exchange rates (~900 s^-1 and ~3600 s^-1).
• The experiment characterized a fast-folding domain with an unfolded state lifetime of ~80 µs.

Conclusions:

• The methyl double quantum coherence experiment is a valuable addition to the toolkit for studying protein dynamics.
• This technique provides accurate measurements of protein exchange rates, expanding the accessible timescale for CPMG relaxation dispersion studies.
• The method offers enhanced insights into the mechanisms of protein conformational changes and function.