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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.

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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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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.
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Using Solution NMR to Characterize Biomolecular Condensates Under Biphasic Conditions.

Mihajlo Novakovic¹, Johannes Schmoll¹, Leonidas Emmanouilidis¹

¹Department of Biology, Institute of Biochemistry, ETH Zurich.

Journal of Visualized Experiments : Jove

|May 4, 2026

Summary

This summary is machine-generated.

Nuclear magnetic resonance (NMR) spectroscopy offers label-free methods to study biomolecular condensates. New NMR techniques, REDIFINE and CONDENSE-MT, characterize condensate dynamics and composition without tags.

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Area of Science:

Biophysics
Molecular Biology
Biochemistry

Background:

Biomolecular condensates, formed via liquid-liquid phase separation (LLPS), are crucial for cellular organization and biochemical regulation.
Investigating condensate composition, dynamics, and internal structure without external tags presents significant challenges.

Purpose of the Study:

To develop and validate novel Nuclear Magnetic Resonance (NMR) methodologies for label-free characterization of biomolecular condensates.
To provide quantitative insights into the physicochemical properties and dynamics of condensates in their native biphasic state.

Main Methods:

Utilized two complementary NMR approaches: REstricted DIffusion of INvisible speciEs (REDIFINE) for dynamic condensates and CONdensate DEtectioN by SEmi-solid Magnetization Transfer (CONDENSE-MT) for rigid condensates.
REDIFINE quantifies phase partitioning, droplet size, interface permeability, and molecular exchange rates using diffusion-exchange contrast.
CONDENSE-MT employs water-detected magnetization transfer to assess partitioning, molecular tumbling, hydration, and bound water in NMR-invisible condensates.

Main Results:

Demonstrated the capability of REDIFINE to measure exchange rates and droplet properties for dynamic condensates.
Showcased CONDENSE-MT's effectiveness in characterizing the biophysical properties of rigid condensates.
Integrated both methods to provide a comprehensive, multidimensional view of condensate structure and dynamics under near-native conditions.

Conclusions:

These advanced NMR techniques offer powerful, label-free tools for studying biomolecular phase separation.
The developed methodologies enable a deeper understanding of condensate physicochemical properties and their link to biological functions and diseases.
Expanded the NMR toolbox for investigating complex cellular organization driven by phase separation.