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Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels
Published on: July 4, 2016
Nanometer-scale distance measurements in proteins using Gd3+ spin labeling.
Alexey Potapov1, Hiromasa Yagi, Thomas Huber
1Department of Chemical Physics, Weizmann Institute of Science, Rehovot, 76100, Israel.
Journal of the American Chemical Society
|June 12, 2010
Summary
We introduce Gadolinium(3+) (Gd3+) spin labeling for measuring nanometer distances in proteins using high-field electron paramagnetic resonance (EPR). Gd3+ labeling offers improved sensitivity and avoids orientation selection issues, making it a viable technique for protein structure analysis.
Area of Science:
- Biophysics
- Structural Biology
- Spectroscopy
Background:
- Accurate measurement of nanometer-scale distances within proteins is crucial for understanding their structure and function.
- Conventional site-directed spin labeling using nitroxides with pulse Electron Paramagnetic Resonance (EPR) is a powerful technique, but can be limited by sensitivity and orientation selection.
Purpose of the Study:
- To introduce and evaluate Gadolinium(3+) (Gd3+) spin labeling as a method for nanometer-range distance measurements in proteins.
- To compare the performance of Gd3+ labeling with traditional nitroxide labeling using double-electron electron resonance (DEER) at various magnetic field strengths (X-band and W-band).
Main Methods:
- Developed Gd3+ spin labels derived from dipicolinic acid, covalently attached to cysteine residues in proteins p75ICD and tau(C)14.
- Performed four-pulse double-electron electron resonance (DEER) measurements at X-band (approx. 9.5 GHz) and W-band (95 GHz).
- Compared distance distributions obtained from proteins labeled with Gd3+ or nitroxide spin labels.
Main Results:
- Gd3+ labeling provided distance distributions in good agreement with structural models, with maxima at 2.9 nm (p75ICD) and 3.4 nm (tau(C)14).
- In p75ICD, nitroxide labeling yielded a distance distribution peaking at 2.5 nm, while Gd3+ labeling showed a peak at 2.9 nm.
- In tau(C)14, W-band nitroxide measurements suffered from orientation selection, whereas Gd3+ labeling at W-band provided clear distance information (peak at 3.4 nm).
Conclusions:
- Gd3+ spin labeling is a viable technique for nanometer-scale distance measurements in proteins using high-field pulse EPR.
- Gd3+ labeling offers an order of magnitude improvement in sensitivity compared to X-band nitroxide EPR.
- A key advantage of Gd3+ labeling is the intrinsic absence of orientation selection, simplifying distance distribution analysis, especially at high fields.
