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15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the µs-ms Timescale
Published on: April 19, 2021
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Off-resonance rotating-frame relaxation dispersion experiment for 13C in aromatic side chains using L-optimized
Ulrich Weininger1, Ulrika Brath, Kristofer Modig
1Department of Biophysical Chemistry, Center for Molecular Protein Science, Lund University, P.O. Box 124, 22100, Lund, Sweden.
Journal of Biomolecular NMR
|April 8, 2014
Summary
This study introduces a new NMR experiment to measure fast protein dynamics in aromatic side chains. The method enhances sensitivity and accuracy for studying protein folding and function.
Area of Science:
- Biophysics
- Structural Biology
- Biochemistry
Background:
- Protein dynamics on microsecond-millisecond timescales are crucial for biological functions like enzyme catalysis and protein folding.
- NMR relaxation dispersion experiments offer site-specific insights into these dynamics.
- Aromatic side chains are important probes due to their roles in binding interfaces, catalysis, and protein structure, but have been less studied.
Purpose of the Study:
- To develop a novel Nuclear Magnetic Resonance (NMR) experiment for measuring microsecond-to-millisecond conformational exchange in aromatic side chains.
- To enhance sensitivity and accuracy in studying protein dynamics using selectively (13)C labeled proteins.
- To extend the repertoire of NMR methods for probing faster dynamic processes in proteins.
Main Methods:
- Development of an off-resonance R 1ρ experiment utilizing longitudinal- and transverse-relaxation optimization.
- Application of selective excitation and inversion of the (13)C doublet for sensitivity enhancement.
- Optimization of longitudinal relaxation recovery of attached (1)H spins for further signal improvement.
- Validation using L-TROSY-selected R 1ρ experiment on Y23 in bovine pancreatic trypsin inhibitor.
Main Results:
- The developed experiment successfully measured exchange parameters for Y23 in bovine pancreatic trypsin inhibitor, with a mean waiting time of 320 μs.
- The determined chemical shift difference accurately matched values obtained from lower-temperature NMR spectra.
- A method was provided to account for complicating effects of strong scalar coupling between protons.
- The experiment demonstrated improved sensitivity and control over artifacts.
Conclusions:
- The new off-resonance R 1ρ experiment effectively measures microsecond-millisecond conformational exchange in aromatic side chains.
- This method provides enhanced sensitivity and accuracy for studying protein dynamics, particularly faster processes.
- The approach extends NMR capabilities for investigating the functional roles of aromatic side chains in proteins.
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