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Videos de Conceptos Relacionados

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

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

1.3K
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...
1.3K
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.
1.4K
¹³C NMR: ¹H–¹³C Decoupling01:04

¹³C NMR: ¹H–¹³C Decoupling

1.7K
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

1.5K
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...
1.5K
Double Resonance Techniques: Overview01:12

Double Resonance Techniques: Overview

870
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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Video Experimental Relacionado

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 Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

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Un protocolo para regular la separación de fases líquido-líquido de proteínas mediante mutagénesis guiada por RMN

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
Resumen

Los investigadores desarrollaron un nuevo método que vincula la estructura de las proteínas con la separación de fases líquido-líquido (LLPS). Esta técnica utiliza resonancia magnética nuclear (RMN) y mutagénesis para controlar el comportamiento de las proteínas, ofreciendo información sobre la formación de compartimentos celulares.

Palabras clave:
mutagénesis guiada por RMNVAPBseparación de fases líquido-líquidoresonancia magnética nuclearcondensados de proteínasdinámica de proteínas

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Área de la Ciencia:

  • Bioquímica y Biología Molecular
  • Biología Celular
  • Biofísica

Sus antecedentes:

  • Los compartimentos celulares sin membrana se forman a través de la separación de fases líquido-líquido (LLPS).
  • Los métodos experimentales que vinculan el comportamiento cuantitativo de LLPS con datos estructurales a nivel de residuo son limitados.
  • La comprensión de la base molecular de LLPS es crucial para la biología celular.

Objetivo del estudio:

  • Desarrollar un protocolo integrado que conecte ensayos cuantitativos de LLPS con información estructural a nivel de residuo.
  • Permitir la regulación de la separación de fases de proteínas a través de mutagénesis guiada por la estructura.
  • Proporcionar un marco generalizable para el control sistemático a nivel de residuo de la LLPS de proteínas.

Principales métodos:

  • Se combinaron ensayos cuantitativos de LLPS con espectroscopia de resonancia magnética nuclear (RMN).
  • Se empleó mutagénesis guiada por la estructura para modificar secuencias de proteínas.
  • Se utilizó el dominio MSP de VAPB como sistema modelo.

Principales resultados:

  • Se vinculó con éxito características estructurales específicas de residuos con el comportamiento macroscópico de LLPS.
  • Se demostró la supresión y mejora de la separación de fases de proteínas mediante sustituciones de aminoácidos dirigidas.
  • Se validó el dominio MSP de VAPB como modelo para estudiar la regulación de LLPS.

Conclusiones:

  • El protocolo desarrollado proporciona una herramienta poderosa para diseccionar los determinantes moleculares de LLPS.
  • Este marco permite la manipulación precisa a nivel de residuo de la separación de fases de proteínas.
  • Los hallazgos ofrecen nuevas vías para comprender y controlar la formación de compartimentos celulares.