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Related Concept Videos

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...
¹H NMR of Conformationally Flexible Molecules: Variable-Temperature NMR01:15

¹H NMR of Conformationally Flexible Molecules: Variable-Temperature NMR

The axial and equatorial protons in cyclohexane can be distinguished by performing a variable-temperature NMR experiment. In this process, except for one proton, the remaining eleven protons are replaced by deuterium. The deuterium substitution avoids the possible peak splitting caused by the spin-spin coupling between the adjacent protons. The remaining proton flips between the axial and equatorial positions.
2D NMR: Homonuclear Correlation Spectroscopy (COSY)01:06

2D NMR: Homonuclear Correlation Spectroscopy (COSY)

Homonuclear correlation spectroscopy, or COSY, is a 2-dimensional NMR technique that provides information about coupled protons. Typically, the geminal and vicinal coupling are observed. For example, consider the COSY spectrum of ethyl acetate, where its 1D proton NMR spectrum is plotted along the vertical and horizontal axes with their corresponding chemical shift scale. Three spots on the diagonal corresponding to the three peaks in the 1D proton spectrum are called diagonal peaks. The COSY...
¹³C NMR: Distortionless Enhancement by Polarization Transfer (DEPT)01:20

¹³C NMR: Distortionless Enhancement by Polarization Transfer (DEPT)

When proton-coupled carbon-13 spectra are simplified by a broadband proton decoupling technique, structural information about the coupled protons is lost. Distortionless enhancement by polarization transfer (DEPT) is a technique that provides information on the number of hydrogens attached to each carbon in a molecule. While the DEPT experiment utilizes complex pulse sequences, the pulse delay and flip angle are specifically manipulated. The resulting signals have different phases depending on...
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
14:55

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Published on: September 17, 2017

Assignment-free solution NMR method reveals CesT as an unswapped homodimer.

Sigrun Rumpel1, Raghavendran Lakshmi, Stefan Becker

  • 1Department for NMR-Based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.

Protein Science : a Publication of the Protein Society
|August 9, 2008
PubMed
Summary

The study reveals the unswapped CesT chaperone dimer in solution using nuclear magnetic resonance. This finding is crucial for understanding effector translocation mechanisms in type three secretion systems (TTSS).

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Area of Science:

  • Structural biology
  • Molecular mechanisms of protein secretion
  • Biophysics

Background:

  • The homodimeric chaperone CesT is unique among type three secretion system (TTSS) chaperones due to its domain-swapped X-ray structure.
  • This domain swap is hypothesized to be important for the mechanism of effector translocation via the TTSS.

Purpose of the Study:

  • To investigate the solution structure of the CesT dimer.
  • To determine if the domain-swapped or an unswapped conformation is present in solution.
  • To develop efficient methods for structural state discrimination.

Main Methods:

  • Utilized two nuclear magnetic resonance (NMR) strategies.
  • Employed pre-existing structural models and residual dipolar couplings (RDCs).
  • Integrated HN-RDCs and carbonyl backbone chemical shifts for simultaneous backbone assignment and structural state discrimination.

Main Results:

  • Demonstrated the presence of the unswapped 35.4-kDa CesT dimer in solution.
  • Developed an efficient method to discriminate between swapped and unswapped structural states.
  • Showcased the utility of HN-RDCs and chemical shifts for rapid structural model differentiation.

Conclusions:

  • The CesT dimer exists in an unswapped state in solution, challenging previous assumptions based on X-ray structures.
  • The presented NMR approach offers a rapid and generalizable method for distinguishing between alternative structural models.
  • This work provides insights into the conformational dynamics of TTSS chaperones and their role in protein translocation.