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¹H NMR: Complex Splitting01:13

¹H NMR: Complex Splitting

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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...
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¹³C NMR: Distortionless Enhancement by Polarization Transfer (DEPT)01:20

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

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

1.4K
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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¹³C NMR: ¹H–¹³C Decoupling01:04

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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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Chemical Shift: Internal References and Solvent Effects01:17

Chemical Shift: Internal References and Solvent Effects

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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.
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Double Resonance Techniques: Overview01:12

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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...
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Updated: May 3, 2026

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
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A Protocol for Regulating Protein Liquid-Liquid Phase Separation Using NMR-Guided Mutagenesis.

Mayu Enomoto-Kusano1, Kyoko Furuita2,3, Takashi S Kodama2

  • 1Graduate School of Engineering Science, Yokohama National University, Tokiwadai 79-5, Hodogaya-ku, Yokohama 240-8501, Kanagawa, Japan.

Methods and Protocols
|February 20, 2026
PubMed
Summary
This summary is machine-generated.

Researchers developed a new method linking protein structure to liquid-liquid phase separation (LLPS). This technique uses nuclear magnetic resonance (NMR) and mutagenesis to control protein behavior, offering insights into cellular compartment formation.

Keywords:
NMR-guided mutagenesisVAPBliquid–liquid phase separationnuclear magnetic resonanceprotein condensatesprotein dynamics

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Area of Science:

  • Biochemistry and Molecular Biology
  • Cell Biology
  • Biophysics

Background:

  • Membraneless cellular compartments form via liquid-liquid phase separation (LLPS).
  • Experimental methods linking quantitative LLPS behavior to residue-level structural data are limited.
  • Understanding the molecular basis of LLPS is crucial for cell biology.

Purpose of the Study:

  • To develop an integrated protocol connecting quantitative LLPS assays with residue-level structural information.
  • To enable regulation of protein phase separation through structure-guided mutagenesis.
  • To provide a generalizable framework for systematic, residue-level control of protein LLPS.

Main Methods:

  • Combined quantitative LLPS assays with nuclear magnetic resonance (NMR) spectroscopy.
  • Employed structure-guided mutagenesis to modify protein sequences.
  • Utilized the VAPB MSP domain as a model system.

Main Results:

  • Successfully linked residue-specific structural features to macroscopic LLPS behavior.
  • Demonstrated suppression and enhancement of protein phase separation via targeted amino acid substitutions.
  • Validated the VAPB MSP domain as a model for studying LLPS regulation.

Conclusions:

  • The developed protocol provides a powerful tool for dissecting the molecular determinants of LLPS.
  • This framework allows for precise, residue-level manipulation of protein phase separation.
  • The findings offer new avenues for understanding and controlling cellular compartment formation.