Multi-probe relaxation dispersion measurements increase sensitivity to protein dynamics

Area of Science:

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

Background:

• Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion is key for studying micro-millisecond protein dynamics.
• Nitrogen-15 ((15)N) relaxation dispersion is established, but studies using other nuclei are less common.
• Understanding protein dynamics is crucial for characterizing functional mechanisms and structural transitions.

Purpose of the Study:

• To report (15)N and proton-1H R2 relaxation dispersion measurements on a dihydrofolate reductase mutant.
• To demonstrate the complementary nature of (15)N and (1)H nuclei for probing protein dynamics.
• To showcase the benefits of simultaneous fitting for accurate determination of exchange parameters.

Main Methods:

• Utilized Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments.
• Performed measurements on both (15)N and (1)H nuclei for the N23PP/S148A mutant of dihydrofolate reductase.
• Employed simultaneous fitting of relaxation dispersion profiles from both nuclei.

Main Results:

• (1)H relaxation dispersion identified dynamics in additional residues compared to (15)N.
• Simultaneous fitting improved the accuracy of exchange parameters for residues and residue clusters.
• Observed hydrogen bond effects, ring current modulations, and backbone torsional changes, providing detailed insights into structural transitions.

Conclusions:

• Combined (15)N and (1)H relaxation dispersion provides a powerful approach for studying micro-millisecond timescale backbone dynamics.
• The differential sensitivity of (15)N and (1)H nuclei offers unique insights into the nature of protein structural changes.
• Analysis of chemical shift changes alongside relaxation dispersion data enhances the understanding of dynamic processes.