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Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels
Published on: July 4, 2016
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High-spin Metal Centres in Dipolar EPR Spectroscopy
Katharina Keller1, Thomas Wiegand1, Riccardo Cadalbert1
1ETH Zurich, Laboratorium für Physikalische Chemie, Vladimir-Prelog-Weg 2, CH-8093 Zurich, Switzerland.
Chimia
|May 4, 2018
Summary
Researchers used manganese (Mn2+) to study the DnaB helicase structure using electron paramagnetic resonance (EPR) spectroscopy. This method allows monitoring of nucleotide binding and probing of protein assembly geometry.
Area of Science:
- Biophysics
- Structural Biology
- Spectroscopy
Background:
- The bacterial DnaB helicase is a crucial ATP:Mg2+-dependent protein engine involved in DNA replication.
- Studying the DnaB helicase's structure and function is essential for understanding DNA replication mechanisms.
- Electron paramagnetic resonance (EPR) spectroscopy is a powerful technique for probing paramagnetic centers in biological systems.
Purpose of the Study:
- To investigate the structural and functional properties of the Helicobacter pylori DnaB helicase.
- To apply electron paramagnetic resonance (EPR) spectroscopy, utilizing Mn2+ substitution for Mg2+, to monitor nucleotide binding.
- To determine the geometry of the multimeric DnaB helicase assembly using double electron-electron resonance (DEER) and relaxation induced dipolar modulation enhancement (RIDME) techniques.
Main Methods:
- Substitution of Mg2+ with Mn2+ in the DnaB helicase to enable EPR spectroscopy.
- Utilizing EPR relaxation measurements to estimate Mn2+ binding fractions and monitor nucleotide interactions.
- Employing ultra-wideband double electron-electron resonance (DEER) and relaxation induced dipolar modulation enhancement (RIDME) for spin-spin distance measurements.
- Applying Tikhonov regularization with a modified kernel function to correct for dipolar frequency overtones in RIDME data analysis.
Main Results:
- Successful application of Mn2+-substituted DnaB helicase for EPR studies, enabling monitoring of nucleotide binding.
- Estimation of Mn2+ binding fractions through EPR relaxation measurements.
- Determination of spin-spin distances within the multimeric helicase assembly using DEER and RIDME.
- Development and validation of a method to correct for dipolar frequency overtones in RIDME analysis for high-spin centers like Mn2+.
Conclusions:
- Mn2+ substitution is a viable strategy for EPR studies of the DnaB helicase, providing insights into nucleotide binding.
- DEER and RIDME are effective for probing the geometry of multimeric protein assemblies like DnaB helicase.
- The developed correction method for dipolar overtones in RIDME analysis is robust and applicable to Mn2+-containing systems.
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