Related Experiment Video
Updated: Jul 21, 2025
11:19
Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels
Published on: July 4, 2016
10.7K
Spectroscopically Orthogonal Spin Labels in Structural Biology at Physiological Temperatures
Markus Teucher1, Svetlana Kucher1,2, M Hadi Timachi1
1Faculty of Chemistry and Biochemistry, Ruhr University of Bochum, Bochum 44801, Germany.
The Journal of Physical Chemistry. B
|July 25, 2023
Summary
This study uses orthogonal spin labels like gadolinium and nitroxide with electron paramagnetic resonance (EPR) spectroscopy to reveal biomolecular dynamics and interactions at physiological temperatures, enhancing structural biology insights.
Area of Science:
- Biophysics
- Structural Biology
- Spectroscopy
Background:
- Electron paramagnetic resonance (EPR) spectroscopy is key for measuring distances in biomacromolecules using pulsed dipolar spectroscopy (PDS) at low temperatures.
- Orthogonal spin labels enhance data acquisition per sample in EPR studies.
- Current EPR methods are often limited to cryogenic temperatures, restricting the study of dynamic processes under physiological conditions.
Purpose of the Study:
- To investigate biomolecular dynamics and interactions at physiological temperatures using EPR.
- To leverage the distinct properties of gadolinium and nitroxide spin labels for enhanced information retrieval.
- To explore side chain and water dynamics, as well as short-range distances in biomolecules under native-like conditions.
Main Methods:
- Utilizing continuous wave (cw) EPR at X band to analyze side chain dynamics.
- Employing Overhauser dynamic nuclear polarization (ODNP) at X band to study surface water dynamics.
- Applying cw EPR at high fields to determine short-range distances.
- Integrating orthogonal spin labels (gadolinium and nitroxide) for multi-modal analysis.
Main Results:
- Demonstrated the capability to study side chain dynamics using cw EPR at X band with specific spin labels.
- Successfully monitored surface water dynamics via ODNP at X band.
- Quantified short-range distances using high-field cw EPR.
- Showcased increased information content from orthogonal labels under physiological conditions.
Conclusions:
- Orthogonal spin labels combined with diverse EPR techniques provide unprecedented insights into biomolecular behavior at physiological temperatures.
- These methods reveal molecular interactions and dynamic equilibria previously inaccessible at cryogenic temperatures.
- The presented approaches significantly advance the study of biomolecules in biologically relevant states.
Related Concept Videos
01:14Atomic Nuclei: Nuclear Spin State Population Distribution
1.0K
Near absolute zero temperatures, in the presence of a magnetic field, the majority of nuclei prefer the lower energy spin-up state to the higher energy spin-down state. As temperatures increase, the energy from thermal collisions distributes the spins more equally between the two states. The Boltzmann distribution equation gives the ratio of the number of spins predicted in the spin −½ (N−) and spin +½ (N+) states.
1.0K
01:08NMR Spectroscopy: Spin–Spin Coupling
1.5K
The spin state of an NMR-active nucleus can have a slight effect on its immediate electronic environment. This effect propagates through the intervening bonds and affects the electronic environments of NMR-active nuclei up to three bonds away; occasionally, even farther. This phenomenon is called spin–spin coupling or J-coupling. Coupling interactions are mutual and result in small changes in the absorption frequencies of both nuclei involved. While nuclei of the same element are involved...
1.5K
01:02¹H NMR: Interpreting Distorted and Overlapping Signals
1.1K
Spin systems where the difference in chemical shifts of the coupled nuclei is greater than ten times J are called first-order spin systems. These nuclei are weakly coupled, and their chemical shifts and coupling constant can generally be estimated from the well-separated signals in the spectrum.
As Δν decreases and the signals move closer, the doublets appear increasingly distorted. The intensities of the inner lines increase at the cost of those of the outer lines as the signals are...
As Δν decreases and the signals move closer, the doublets appear increasingly distorted. The intensities of the inner lines increase at the cost of those of the outer lines as the signals are...
1.1K
01:03Atomic Nuclei: Nuclear Spin State Overview
1.0K
NMR-active nuclei have energy levels called 'spin states' that are associated with the orientations of their nuclear magnetic moments. In the absence of a magnetic field, the nuclear magnetic moments are randomly oriented, and the spin states are degenerate. When an external magnetic field is applied, the spin states have only 2 + 1 orientations available to them. A proton with = ½ has two available orientations. Similarly, for a quadrupolar nucleus with a nuclear spin value of...
1.0K
01:15¹H NMR of Conformationally Flexible Molecules: Variable-Temperature NMR
1.1K
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.
1.1K
01:162D NMR: Overview of Homonuclear Correlation Techniques
239
Homonuclear correlation spectroscopy (COSY) is a powerful technique used in Nuclear Magnetic Resonance (NMR) spectroscopy to study the correlations between nuclei of the same type within a molecule. It provides information about scalar couplings between adjacent nuclei, which helps determine connectivity and structural information. There are several COSY variants, each with its unique strengths and experimental parameters.
COSY90 is the standard two-dimensional (2D) COSY experiment that...
COSY90 is the standard two-dimensional (2D) COSY experiment that...
239