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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
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Simultaneous Recording of Remote Domain Dynamics in Membrane Proteins Using the Double-Labeled DXB/DXT Technique.

Kazuhiro Mio1,2, Tatsunari Ohkubo1,2, Daisuke Sasaki3

  • 1AIST-UTokyo Advanced Operando-Measurement Technology Open Innovation Laboratory (OPERANDO-OIL), National Institute of Advanced Industrial Science and Technology (AIST), 6-2-3 Kashiwanoha, Chiba 277-0882, Japan.

Membranes
|April 26, 2024
PubMed
Summary

This study introduces dual-labeling for diffracted X-ray blinking (DXB) and tracking (DXT) to simultaneously observe protein domain motions. This technique reveals ligand-induced dynamics in receptors like 5-HT2A and TRPV1.

Keywords:
diffracted X-ray blinking (DXB)diffracted X-ray tracking (DXT)dual-labeling techniqueintramolecular dynamicsmembrane proteins

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Area of Science:

  • Biophysics
  • Structural Biology
  • Biochemistry

Background:

  • Protein dynamics are crucial for biological functions and allosteric regulation.
  • Existing methods like Diffracted X-ray Blinking (DXB) and Diffracted X-ray Tracking (DXT) analyze protein motion using nanocrystal probes.
  • Simultaneous observation of motions across different protein domains has been a challenge.

Purpose of the Study:

  • To develop a dual-labeling technique for simultaneous DXB and DXT analysis of protein dynamics.
  • To validate zinc oxide (ZnO) crystals as effective secondary labeling probes.
  • To investigate ligand-induced conformational changes in the 5-HT2A and TRPV1 receptors.

Main Methods:

  • Developed a dual-labeling strategy for DXB and DXT using gold and zinc oxide (ZnO) nanocrystals.
  • Utilized ZnO crystals for their diffraction properties, stability, and protein binding.
  • Applied dual-labeling DXB to study the 5-HT2A receptor in living cells.
  • Applied dual-labeling DXT to analyze capsaicin-induced motions in the TRPV1 protein.

Main Results:

  • Simultaneous motion analysis of the N-terminus and extracellular loop of the 5-HT2A receptor showed ligand-induced motion suppression.
  • Dual-labeling DXT revealed a capsaicin-induced peak shift in the N-terminus of TRPV1, but not the C-terminus.
  • Capsaicin-induced motion modulation in TRPV1 was reversible with a competitive inhibitor (AMG9810).

Conclusions:

  • The dual-labeling DXB and DXT technique enables simultaneous observation of protein dynamics at multiple sites.
  • This method provides insights into ligand-receptor interactions and allosteric mechanisms.
  • The findings highlight the utility of ZnO crystals as secondary probes for advanced protein dynamics studies.