Rigid and stable nitroxide spin label for high-resolution distance measurements on proteins by DEER experiments

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Pulsed electron-electron double resonance (PELDOR or DEER) measures distances between spin labels in proteins, providing structural restraints.
• Site-specific spin labeling enhances accuracy and precision in distance measurements.

Purpose of the Study:

• To introduce a novel spin labeling approach using a thioester bond for improved DEER measurements.
• To evaluate the performance of the NOAI (NO spin labels activated by acetylimidazole) labeling method.

Main Methods:

• Site-specific protein labeling via thioester bond formation between NOAI and protein thiols.
• DEER distance measurements on labeled proteins, including ubiquitin, GB1, and Smac.
• Comparison of NOAI labeling with existing methods (MTSL, M-Prox).

Main Results:

• NOAI exhibits a shorter connection to the protein ligation site compared to MTSL and M-Prox.
• NOAI labeling resulted in narrower distance distributions for tested proteins.
• NOAI conjugates are stable against reducing agents and enable high-fidelity DEER measurements in cell lysates.

Conclusions:

• The NOAI thioester labeling method provides high-fidelity DEER distance measurements with improved accuracy.
• This method offers a robust tool for protein structural and conformational analysis, even in complex biological samples like cell lysates.