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Related Concept Videos

Double Resonance Techniques: Overview01:12

Double Resonance Techniques: Overview

Double resonance techniques in Nuclear Magnetic Resonance (NMR) spectroscopy involve the simultaneous application of two different frequencies or radiofrequency pulses to manipulate and observe two distinct nuclear spins. One important application of double resonance is spin decoupling, which selectively suppresses coupling with one type of nucleus while observing the NMR signal from another nucleus, simplifying the spectrum and enhancing resolution.
Spin decoupling is usually achieved by...
Chemical Shift: Internal References and Solvent Effects01:17

Chemical Shift: Internal References and Solvent Effects

In an NMR sample, precise measurement of the absolute absorption frequencies of nuclei is difficult. A standard internal reference compound is added, and the frequency difference between the reference signal and sample signals is measured.
The internal reference compound generally used in NMR spectroscopy is tetramethylsilane (TMS). TMS is preferred because it is chemically inert, soluble in NMR solvents, and easily removable. Also, the highly shielded methyl protons in TMS yield an intense...
¹H NMR: Complex Splitting01:13

¹H NMR: Complex Splitting

A proton M that is coupled to a proton X results in doublet signals for M. However, NMR-active nuclei can be simultaneously coupled to more than one nonequivalent nucleus. When M is coupled to a second proton A, such as in styrene oxide, each peak in the doublet is split into another doublet.
Splitting diagrams or splitting tree diagrams are routinely used to depict such complex couplings. While drawing splitting diagrams, the splitting with the larger coupling constant is usually applied first.
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 and Mass Spectroscopy of Carboxylic Acids01:30

NMR and Mass Spectroscopy of Carboxylic Acids

In ¹H NMR spectroscopy, acidic protons (–COOH) of carboxylic acids are highly deshielded and absorb far downfield, at around 9–12 ppm. The chemical shift value depends on the concentration and solvent used.
While α protons of carboxylic acids absorb at 2–2.5 ppm, β protons absorb further upfield.
Carboxylic acids are easily identified by dissolving them in deuterium oxide, which results in a rapid exchange of the acidic protons with deuterium. This leads to the disappearance of the acidic...
¹H NMR of Conformationally Flexible Molecules: Temporal Resolution00:52

¹H NMR of Conformationally Flexible Molecules: Temporal Resolution

At room temperature, the chair conformer of cyclohexane undergoes rapid ring flipping between two equivalent chair conformers at a rate of approximately 105 times per second. These two chair conformers are in equilibrium. The rapid ring flipping results in the interconversion of the axial proton to an equatorial proton and an equatorial to the axial proton. Such interconversions are too rapid and cannot be detected on the NMR timescale. Hence, the NMR spectrometer cannot distinguish between the...

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Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy
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Direct methods and residue type specific isotope labeling in NMR structure determination and model-driven sequential

Andreas Schedlbauer1, Renate Auer, Karin Ledolter

  • 1Institute of Biomolecular Structural Chemistry, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5/1, Vienna, Austria.

Journal of Biomolecular NMR
|September 3, 2008
PubMed
Summary

This study presents a hybrid approach for protein structure determination using Nuclear Magnetic Resonance (NMR). It enhances sequential backbone assignment by integrating low-resolution models with Nuclear Overhauser Effect (NOE) data, even with incomplete information.

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Published on: December 12, 2013

Area of Science:

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

Background:

  • Direct NMR methods for protein structure determination typically require extensive Nuclear Overhauser Effect (NOE) data for ab initio structure solving.
  • Low-resolution protein models can be generated with fewer restraints but may lack stereochemical definition, making it difficult to distinguish correct folds from mirror images.
  • Standard NMR assignment strategies can be limited by sensitivity issues, particularly when (13)C(beta) and (13)C' chemical shifts are unavailable.

Purpose of the Study:

  • To develop a hybrid NMR approach that leverages low-resolution structural models to improve sequential backbone chemical shift assignment.
  • To enhance the information content of NOE spectra by integrating ambiguous structural models.
  • To assess the utility of residue-specific labeling and minimal triple-resonance data in conjunction with this hybrid method.

Main Methods:

  • Generation of low-resolution protein models from limited distance restraints derived from NOE data.
  • Integration of these ambiguous models with NOE spectra to aid sequential backbone assignment.
  • Application of residue-specific isotope labeling and minimal triple-resonance experiments (e.g., using (13)C(alpha) connectivity).

Main Results:

  • The hybrid approach enhances the information content of NOE spectra, facilitating sequential assignment.
  • This method provides significant improvements in assignment accuracy, even when (13)C(beta) and (13)C' shifts are not available.
  • The strategy demonstrates tolerance to data incompleteness, imprecise peak picking, and potential NOE misassignments.

Conclusions:

  • A hybrid NMR strategy combining low-resolution models with NOE data effectively aids sequential backbone assignment.
  • Customized isotope labeling patterns are advantageous for this approach.
  • The method offers a robust alternative for protein structure determination, particularly under challenging data conditions.