Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

¹H NMR of Conformationally Flexible Molecules: Temporal Resolution00:52

¹H NMR of Conformationally Flexible Molecules: Temporal Resolution

966
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...
966
¹H NMR of Conformationally Flexible Molecules: Variable-Temperature NMR01:15

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

1.3K
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.
1.3K
IR Frequency Region: Fingerprint Region01:03

IR Frequency Region: Fingerprint Region

1.4K
IR spectra are divided into two main regions: the diagnostic region and the fingerprint region. The diagnostic region of the spectrum lies above 1500 cm−1. The absorptions resulting from single-bond vibrations of the N–H, C–H, and O–H stretch at higher wavenumbers and appear on the left side of the spectrum. The stretching absorptions of the C≡C and C≡N occur between 2100–2300 cm−1. In contrast, those arising from stretching absorptions of the...
1.4K

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Biochemical Consequences of a Leucine-to-Cysteine Clamp Substitution in Lipoxygenases.

Biomolecules·2025
Same author

Impact of <i>N</i>-Glycosylation on Protein Structure and Dynamics Linked to Enzymatic C-H Activation in the <i>M. oryzae</i> Lipoxygenase.

Biochemistry·2024
Same author

Identification of the Thermal Activation Network in Human 15-Lipoxygenase-2: Divergence from Plant Orthologs and Its Relationship to Hydrogen Tunneling Activation Barriers.

ACS catalysis·2024
Same author

<sup>13</sup>C Electron Nuclear Double Resonance Spectroscopy-Guided Molecular Dynamics Computations Reveal the Structure of the Enzyme-Substrate Complex of an Active, <i>N</i>-Linked Glycosylated Lipoxygenase.

Biochemistry·2023
Same author

Electrochemical and Structural Study of the Buried Tryptophan in Azurin: Effects of Hydration and Polarity on the Redox Potential of W48.

The journal of physical chemistry. B·2022
Same author

Membrane-Mimicking Reverse Micelles for High-Resolution Interfacial Study of Proteins and Membranes.

Langmuir : the ACS journal of surfaces and colloids·2022

Related Experiment Video

Updated: Oct 27, 2025

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy
10:03

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Published on: June 27, 2014

18.1K

Detection of Catalytically Linked Conformational Changes in Wild-Type Class Ia Ribonucleotide Reductase Using

Ryan Atlee Watson1, Adam R Offenbacher1,2, Bridgette A Barry1

  • 1Department of Chemistry and Biochemistry and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, United States.

The Journal of Physical Chemistry. B
|July 22, 2021
PubMed
Summary

Ribonucleotide reductase (RNR) is crucial for DNA synthesis. A new RIFTIR spectroscopy method reveals conformational changes during RNR catalysis, including a key tyrosine in the PCET pathway.

More Related Videos

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase
10:01

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Published on: December 4, 2017

12.4K
Practical Aspects of Sample Preparation and Setup of 1H R1&#961; Relaxation Dispersion Experiments of RNA
08:17

Practical Aspects of Sample Preparation and Setup of 1H R1ρ Relaxation Dispersion Experiments of RNA

Published on: July 9, 2021

4.9K

Related Experiment Videos

Last Updated: Oct 27, 2025

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy
10:03

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Published on: June 27, 2014

18.1K
Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase
10:01

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Published on: December 4, 2017

12.4K
Practical Aspects of Sample Preparation and Setup of 1H R1&#961; Relaxation Dispersion Experiments of RNA
08:17

Practical Aspects of Sample Preparation and Setup of 1H R1ρ Relaxation Dispersion Experiments of RNA

Published on: July 9, 2021

4.9K

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Spectroscopy

Background:

  • Ribonucleotide reductase (RNR) is essential for DNA synthesis, catalyzing the conversion of ribonucleotides to deoxyribonucleotides.
  • The class Ia *Escherichia coli* RNR (α₂β₂) is an unstable heterodimer of dimers, making structural and mechanistic studies challenging.
  • A long-range proton-coupled electron-transfer (PCET) pathway involving a diiron cofactor and active site cysteines is critical for RNR catalysis.

Purpose of the Study:

  • To develop and apply a novel reaction-induced Fourier transform infrared (RIFTIR) spectroscopy method to study the active, wild-type RNR α₂β₂ complex.
  • To elucidate conformational changes associated with RNR catalysis, including the roles of carboxylate interactions, deprotonation, and cysteine oxidation.
  • To investigate the function of specific residues, such as Y356β, within the PCET pathway and the effects of inhibitors.

Main Methods:

  • Development of a reaction-induced Fourier transform infrared (RIFTIR) spectroscopy technique.
  • Spectroscopic analysis of the active, wild-type *Escherichia coli* RNR α₂β₂ complex.
  • Site-specific labeling of tyrosine residues to track conformational dynamics.

Main Results:

  • RIFTIR spectroscopy successfully monitored the mechanism of the active RNR α₂β₂ complex, overcoming previous instability issues.
  • Identified conformational changes, including carboxylate interactions, deprotonation, cysteine oxidation, and reversible secondary structural alterations.
  • Discovered a conformationally active tyrosine (Y356β) involved in the intersubunit PCET pathway and observed allosteric effects of inhibitors like azidoUDP and dATP.

Conclusions:

  • RIFTIR spectroscopy is a powerful tool for studying the dynamic mechanism of the wild-type RNR α₂β₂ complex.
  • The study provides new insights into the conformational landscape of RNR during catalysis and the role of Y356β in PCET.
  • The findings enhance understanding of RNR allostery and inhibition mechanisms.