Efficient sampling of molecular orientations for Cu(II)-based DEER on protein labels

Area of Science:

• Biophysics
• Spectroscopy
• Structural Biology

Background:

• Pulsed-electron paramagnetic resonance (EPR) techniques, particularly double electron-electron resonance (DEER), are crucial for measuring distances in proteins using spin labels like Cu(II).
• A significant challenge in DEER is 'orientational selectivity,' where the dipolar signal is biased by unknown relative spin orientations, hindering accurate distance measurements.
• This orientational bias affects other pulsed-EPR methods probing electron-nucleus interactions, limiting their precision.

Purpose of the Study:

• To dissect and mitigate the effects of orientational selectivity in Cu(II)-labeled proteins using double electron-electron resonance (DEER) at Q-band frequencies.
• To optimize pulsed-EPR acquisition schemes for more accurate distance constraint measurements in protein structure and dynamics.
• To develop a protocol for orientational-independent DEER measurements feasible on commercial spectrometers.

Main Methods:

• Generation of an in silico sample of Cu(II)-labeled proteins to analyze pulse excitation and dipolar signal contributions from various protein orientations.
• Incorporation of realistic pulse excitation profiles to identify excited spins and evaluate different pulse shapes (e.g., rectangular pulses).
• Systematic examination of frequency offsets between the two pulses in DEER experiments to determine optimal settings for distance measurements.

Main Results:

• Computational analysis revealed the contribution of each protein orientation to the dipolar signal, providing insights for optimizing acquisition schemes.
• Rectangular pulses were found to sample spin orientations similarly to more complex pulses with the same bandwidth.
• A two-measurement protocol was developed to achieve orientational-independent DEER at Q-band, experimentally validated on two proteins.
• Increasing rectangular pulse amplitude enhanced DEER experiment efficiency by up to threefold.

Conclusions:

• The study presents a novel computational approach to dissect and overcome orientational selectivity in pulsed-EPR, specifically DEER.
• The developed protocol enables accurate, orientational-independent distance measurements using standard commercial EPR spectrometers.
• This work offers a powerful strategy for obtaining detailed microscopic structural and dynamic information from proteins using EPR spectroscopy.