Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected 13C CPMG relaxation dispersion

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Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected 13C CPMG relaxation dispersion


Abstract Protein dynamics on the millisecond time scale commonly reflect conformational transitions between distinct functional states. NMR relaxation dispersion experiments have provided important insights into biologically relevant dynamics with site-specific resolution, primarily targeting the protein backbone and methyl-bearing side chains. Aromatic side chains represent attractive probes of protein dynamics because they are over-represented in protein binding interfaces, play critical roles in enzyme catalysis, and form an important part of the core. Here we introduce a method to characterize millisecond conformational exchange of aromatic side chains in selectively 13C labeled proteins by means of longitudinal- and transverse-relaxation optimized CPMG relaxation dispersion. By monitoring 13C relaxation in a spin-state selective manner, significant sensitivity enhancement can be achieved in terms of both signal intensity and the relative exchange contribution to transverse relaxation. Further signal enhancement results from optimizing the longitudinal relaxation recovery of the covalently attached 1H spins. We validated the L-TROSY-CPMG experiment by measuring fast foldingâ??unfolding kinetics of the small protein CspB under native conditions. The determined unfolding rate matches perfectly with previous results from stopped-flow kinetics. The CPMG-derived chemical shift differences between the folded and unfolded states are in excellent agreement with those obtained by urea-dependent chemical shift analysis. The present method enables characterization of conformational exchange involving aromatic side chains and should serve as a valuable complement to methods developed for other types of protein side chains.

• Content Type Journal Article
• Category Communication
• Pages 1-6
• DOI 10.1007/s10858-012-9656-z
• Authors
  • Ulrich Weininger, Department of Biophysical Chemistry, Center for Molecular Protein Science, Lund University, P.O. Box 124, 22100 Lund, Sweden
  • Michal Respondek, Department of Biophysical Chemistry, Center for Molecular Protein Science, Lund University, P.O. Box 124, 22100 Lund, Sweden
  • Mikael Akke, Department of Biophysical Chemistry, Center for Molecular Protein Science, Lund University, P.O. Box 124, 22100 Lund, Sweden

• • Journal Journal of Biomolecular NMR
  • Online ISSN 1573-5001
  • Print ISSN 0925-2738

Source: Journal of Biomolecular NMR