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Related Concept Videos

2D NMR: Heteronuclear Single-Quantum Correlation Spectroscopy (HSQC)01:19

2D NMR: Heteronuclear Single-Quantum Correlation Spectroscopy (HSQC)

Heteronuclear single-quantum correlation spectroscopy (HSQC) is a 2D NMR technique that reveals one-bond correlations between hydrogen and a heteronucleus. The HSQC experiment is similar to the heteronuclear correlation experiment (HETCOR) but is more sensitive. In the HSQC spectrum, the proton chemical shift is plotted on the horizontal F2 axis, while the 13C chemical shift is plotted on the vertical F1 axis. The corresponding proton and 13C spectra are also shown. The HSQC contour plot does...
¹³C NMR: ¹H–¹³C Decoupling01:04

¹³C NMR: ¹H–¹³C Decoupling

The probability of having two carbon-13 atoms next to each other is negligible because of the low natural abundance of carbon-13. Consequently, peak splitting due to carbon-carbon spin-spin coupling is not observed in spectra. However, protons up to three sigma bonds away split the carbon signal according to the n+1 rule, resulting in complicated spectra.
A broadband decoupling technique is used to simplify these complex, sometimes overlapping, signals. Broadband decoupling relies on a...

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Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy
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13C-NOESY-HSQC with Split Carbon Evolution for Increased Resolution with Uniformly Labeled Proteins

Baur1, Gemmecker, Kessler

  • 1Institut für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, Garching, D-85747, Germany

Journal of Magnetic Resonance (San Diego, Calif. : 1997)
|June 20, 1998
PubMed
Summary

New pulse sequences improve resolution in 2D Carbon-13 Heteronuclear Single Quantum Coherence (13C-HSQC) and 3D 13C-NOESY-HSQC experiments. These methods reduce signal loss and peak splitting, enhancing spectral clarity for protein analysis.

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Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Published on: June 8, 2021

Area of Science:

  • Nuclear Magnetic Resonance (NMR) Spectroscopy
  • Protein Structure Determination
  • Biophysical Chemistry

Background:

  • Standard 2D 13C-HSQC and 3D 13C-NOESY-HSQC experiments can suffer from signal loss and peak splitting due to long carbon evolution times.
  • 13C homonuclear J-couplings often limit spectral resolution and sensitivity in these experiments.
  • Improved NMR techniques are crucial for detailed structural analysis of large biomolecules.

Purpose of the Study:

  • To introduce and validate two novel pulse sequences for 2D 13C-HSQC and 3D 13C-NOESY-HSQC experiments.
  • To enhance spectral resolution in the carbon domain while maintaining good sensitivity, even for large proteins.
  • To overcome limitations imposed by 13C homonuclear J-couplings in NMR spectroscopy.

Main Methods:

  • Development of pulse sequences with two consecutive carbon evolution periods separated by a z-filter.
  • Utilizing a non-constant-time incrementation scheme to minimize J-coupling effects.
  • Employing a 13C-13C gradient echo acquisition protocol for data collection.
  • Application to uniformly 13C-labeled proteins, including the IIAMan protein from E. coli.

Main Results:

  • Achieved nearly twofold enhancement in carbon spectral resolution compared to standard 13C-HSQC at twice the evolution time.
  • Demonstrated a 36% decrease in linewidths for the IIAMan protein using the new 13C-HSQC sequence.
  • Observed a reduction in signal-to-noise ratio (S/N) of 40% for representative cross-peaks, consistent with increased resolution.
  • The derived 13C-NOESY-HSQC experiment provided additional Nuclear Overhauser Effect (NOE) restraints for previously unresolved resonances.

Conclusions:

  • The new pulse sequences effectively suppress 13C homonuclear J-couplings, leading to improved spectral resolution and reduced peak splitting.
  • These advanced NMR techniques offer enhanced capabilities for structural studies of uniformly 13C-labeled proteins.
  • The method provides valuable additional NOE restraints, aiding in the detailed structural elucidation of complex biomolecules.