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Related Experiment Video

Updated: Mar 26, 2026

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Published on: November 8, 2019

Detecting O2 binding sites in protein cavities.

Ryo Kitahara¹, Yuichi Yoshimura², Mengjun Xue²

¹College of Pharmaceutical Sciences, Ritsumeikan University, Nojihigashi 1-1-1, Kusatsu 525-8577, Japan.

Scientific Reports

|February 3, 2016

Summary

Researchers used NMR spectroscopy to show that molecular oxygen (O2) binds to hydrophobic cavities in T4 lysozyme. This technique offers a sensitive method for detecting O2 binding in proteins.

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Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Published on: December 28, 2016

Area of Science:

Biochemistry
Structural Biology
Biophysics

Background:

Internal cavities are crucial for protein structure, dynamics, stability, and function.
Understanding ligand binding to these cavities is key to protein mechanism studies.

Purpose of the Study:

To investigate the specific binding of molecular oxygen (O2) to internal cavities in the L99A mutant of T4 lysozyme.
To demonstrate a novel NMR-based method for detecting oxygen binding in proteins.

Main Methods:

Nuclear Magnetic Resonance (NMR) spectroscopy was employed to study O2 binding.
Paramagnetic relaxation effects and chemical shift perturbations were analyzed.
Molecular dynamics simulations were performed to visualize O2 motion and binding pathways.

Main Results:

Specific changes in NMR signals (chemical shifts, broadening) were observed for residues near hydrophobic cavities upon O2 addition.
O2-induced longitudinal relaxation enhancements were consistent with paramagnetic dipolar relaxation.
The study provides the first experimental evidence for O2 binding to hydrophobic, not hydrophilic, cavities in a protein.
Molecular dynamics simulations identified a potential channel for O2 entry and exit.

Conclusions:

Molecular oxygen specifically binds to hydrophobic cavities within T4 lysozyme.
NMR spectroscopy, particularly O2 gas-pressure measurements, is a highly sensitive method for detecting and characterizing oxygen binding sites in proteins.
This technique can detect cavities populated by O2 at concentrations as low as 1%.