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¹H NMR of Conformationally Flexible Molecules: Temporal Resolution

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 NMR

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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.

Cooperative Allosteric Transitions

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Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...

Cooperative Allosteric Transitions

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Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...

Cooperative Allosteric Transitions

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Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...

¹H NMR: Interpreting Distorted and Overlapping Signals

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Spin systems where the difference in chemical shifts of the coupled nuclei is greater than ten times J are called first-order spin systems. These nuclei are weakly coupled, and their chemical shifts and coupling constant can generally be estimated from the well-separated signals in the spectrum.
As Δν decreases and the signals move closer, the doublets appear increasingly distorted. The intensities of the inner lines increase at the cost of those of the outer lines as the signals are slanted or...

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Related Experiment Video

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Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Published on: May 29, 2011

Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR.

Shelley M Elvington¹, Corey W Liu, Merritt C Maduke

¹Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5345, USA.

The EMBO Journal

|September 12, 2009

Summary

Conformational changes in CLC antiporters are not limited to the ion pathway. Fluorine NMR reveals substrate-induced movements at the dimer interface, demonstrating broader protein dynamics during ion transport.

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Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

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Published on: May 29, 2011

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Published on: January 25, 2020

Area of Science:

Biochemistry
Structural Biology
Membrane Protein Dynamics

Background:

The CLC family includes Cl(-) channels and Cl(-)/H(+) antiporters, crucial for ion transport.
While CLC channels exhibit large conformational changes, antiporter movements are less understood, often presumed localized.
The static crystal structure of E. coli ClC-ec1 antiporter lacks dynamic information on ion transport mechanisms.

Purpose of the Study:

To investigate substrate-induced conformational changes in the ClC-ec1 antiporter.
To determine if conformational changes in CLC antiporters are restricted to the ion permeation pathway.
To assess the utility of fluorine NMR for studying membrane protein dynamics.

Main Methods:

Utilized fluorine nuclear magnetic resonance ((19)F NMR) spectroscopy.
Employed mutational analysis to probe protein structure and function.
Monitored substrate-dependent spectral changes to infer conformational movements.

Main Results:

Substrate-dependent (19)F NMR spectral changes were observed in ClC-ec1.
These spectral changes correlate with functionally relevant protein movements at the dimer interface.
Evidence suggests conformational changes extend beyond the Cl(-) permeation pathway.

Conclusions:

Conformational changes in CLC antiporters are not confined to the Cl(-) permeation pathway.
(19)F NMR is a valuable tool for studying dynamic conformational changes in membrane proteins.
Substrate binding induces significant movements within the ClC-ec1 dimer structure.