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Default 13C relaxation experiments for aromatic side chains employing longitudinal- and transverse-relaxation optimized NMR spectroscopy

13C relaxation experiments for aromatic side chains employing longitudinal- and transverse-relaxation optimized NMR spectroscopy


Abstract Aromatic side chains are prevalent in protein binding sites, perform functional roles in enzymatic catalysis, and form an integral part of the hydrophobic core of proteins. Thus, it is of great interest to probe the conformational dynamics of aromatic side chains and its response to biologically relevant events. Indeed, measurements of 13C relaxation rates in aromatic moieties have a long history in biomolecular NMR, primarily in the context of samples without isotope enrichment that avoid complications due to the strong coupling between neighboring 13C spins present in uniformly enriched proteins. Recently established protocols for specific 13C labeling of aromatic side chains enable measurement of 13C relaxation that can be analyzed in a straightforward manner. Here we present longitudinal- and transverse-relaxation optimized pulse sequences for measuring R 1, R 2, and {1H}‚??13C NOE in specifically 13C-labeled aromatic side chains. The optimized R 1 and R 2 experiments offer an increase in sensitivity of up to 35 % for medium-sized proteins, and increasingly greater gains are expected with increasing molecular weight and higher static magnetic field strengths. Our results highlight the importance of controlling the magnetizations of water and aliphatic protons during the relaxation period in order to obtain accurate relaxation rate measurements and achieve full sensitivity enhancement. We further demonstrate that potential complications due to residual two-bond 13C‚??13C scalar couplings or dipolar interactions with neighboring 1H spins do not significantly affect the experiments. The approach presented here should serve as a valuable complement to methods developed for other types of protein side chains.
  • Content Type Journal Article
  • Category Article
  • Pages 1-10
  • DOI 10.1007/s10858-012-9650-5
  • Authors
    • Ulrich Weininger, Center for Molecular Protein Science, Department of Biophysical Chemistry, Lund University, P.O. Box 124, 22100 Lund, Sweden
    • Carl Diehl, Center for Molecular Protein Science, Department of Biophysical Chemistry, Lund University, P.O. Box 124, 22100 Lund, Sweden
    • Mikael Akke, Center for Molecular Protein Science, Department of Biophysical Chemistry, Lund University, P.O. Box 124, 22100 Lund, Sweden

Source: Journal of Biomolecular NMR
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