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Related Concept Videos

NMR Spectroscopy Of Amines01:19

NMR Spectroscopy Of Amines

In proton NMR spectroscopy, primary amines and secondary amines showcase their N–H protons as a broad signal in the chemical shift range between δ 0.5 and 5 ppm. The exact position in this range depends on several factors, including sample concentration, hydrogen bonding, and the type of solvent used. Since amine protons undergo fast proton exchange in solution, the protons are labile and therefore do not participate in any splitting with adjacent protons. Thus, the observed peak is broad and...
2D NMR: Overview of Homonuclear Correlation Techniques01:16

2D NMR: Overview of Homonuclear Correlation Techniques

Homonuclear correlation spectroscopy (COSY) is a powerful technique used in Nuclear Magnetic Resonance (NMR) spectroscopy to study the correlations between nuclei of the same type within a molecule. It provides information about scalar couplings between adjacent nuclei, which helps determine connectivity and structural information. There are several COSY variants, each with its unique strengths and experimental parameters.
COSY90 is the standard two-dimensional (2D) COSY experiment that...
NMR Spectroscopy of Aromatic Compounds01:14

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Aromatic compounds can be identified or analyzed using proton NMR and carbon‐13 NMR. Typically, aromatic hydrogens or hydrogens directly bonded to the aromatic rings are strongly deshielded by the aromatic ring current. Therefore, they absorb in the range of 6.5–8.0 ppm in proton NMR spectra. For instance, aromatic hydrogens directly bonded to the benzene ring absorb at 7.3 ppm. However, aromatic hydrogens of larger rings absorb farther upfield or downfield than the ideal range. Consider...
NMR Spectrometers: Overview01:20

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NMR spectrometers consist of a strong magnet, a radiofrequency transmitter, and a detector attached to a computer console for recording spectra of samples containing NMR-active nuclei. In first-generation NMR instruments called continuous-wave spectrometers, the resonance frequencies of the nuclei are determined by frequency-sweep or field-sweep methods. The magnetic field strength is fixed and the rf signal is swept in the former, while the radiofrequency signal is fixed and the magnetic field...
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Organic molecules primarily contain carbon and hydrogen atoms. While all the hydrogen isotopes are NMR-active, protium or hydrogen-1 is the most abundant. It has a significant energy separation between its nuclear spin states due to its large gyromagnetic ratio. As per Boltzmann's distribution, an increase in the energy separation implies a greater excess population of nuclei available for excitation, resulting in a strong NMR absorption signal.
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Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy
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APSY-NMR with proteins: practical aspects and backbone assignment.

Sebastian Hiller1, Gerhard Wider, Kurt Wüthrich

  • 1Institute of Molecular Biology and Biophysics, ETH Zürich, 8093, Zürich, Switzerland. hiller.sebastian@gmail.com

Journal of Biomolecular NMR
|October 9, 2008
PubMed
Summary
This summary is machine-generated.

Automated projection spectroscopy (APSY) optimizes NMR experiments for protein analysis. This study details setup, sensitivity, and analysis for automated protein backbone assignments, enhancing NMR data interpretation.

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Area of Science:

  • Nuclear Magnetic Resonance (NMR) Spectroscopy
  • Structural Biology
  • Biophysics

Background:

  • Automated projection spectroscopy (APSY) is an NMR technique for recording discrete projection spectra from higher-dimensional NMR experiments.
  • The dedicated algorithm GAPRO automatically identifies multidimensional chemical shift correlations.
  • Sensitivity is a key limiting factor for APSY-NMR applications with larger proteins or dilute samples.

Purpose of the Study:

  • To present technical details for optimizing the setup and analysis of APSY-NMR experiments with proteins.
  • To investigate the effects of varying experimental parameters on sensitivity.
  • To guide the selection of projection angles, sweep widths, and acquisition/processing parameters for APSY-NMR.

Main Methods:

  • Performed a 4D APSY-HNCOCA experiment with a 12-kDa protein at the limit of sensitivity (13 min acquisition time).
  • Generated expressions for APSY-NMR sensitivity based on experimental data and general considerations.
  • Introduced a new peak picking routine and a validation tool for GAPRO spectral analysis.

Main Results:

  • Optimized experimental parameters to enhance sensitivity in APSY-NMR experiments.
  • Developed formulas to guide the selection of projection angles and sweep widths for improved sensitivity.
  • Introduced novel tools for peak picking and validation in GAPRO spectral analysis.

Conclusions:

  • APSY-NMR is a valuable technique for protein structural analysis, with optimized parameters enhancing sensitivity.
  • The developed sensitivity expressions and new analysis tools facilitate automated protein backbone assignments.
  • This work provides a systematic approach for selecting minimal APSY-NMR experiments for automated protein backbone assignment.