Binding Affinity Determines the Success of Endogenous Cu^II-NTA Spin Labeling for In-Cell Electron Paramagnetic...

Area of Science:

• Biophysical Chemistry
• Molecular Biophysics
• Spectroscopy

Background:

• In-cell electron paramagnetic resonance (EPR) spectroscopy enables studying biomolecular structure and dynamics within living cells.
• Genetically encoded double-histidine (dHis) tags offer a platform for endogenous metal-ion-based spin labeling.
• Copper(II)-nitrilotriacetic acid (Cu^II-NTA) coordination is a promising strategy for dHis tag spin labeling.

Purpose of the Study:

• To investigate the impact of varying Cu^II-NTA binding affinities on the success of in-cell EPR experiments.
• To determine the dissociation constants (K_d) for Cu^II-NTA binding to different dHis tag locations.
• To assess the reliability of in-cell EPR techniques, including pulsed electron-electron double resonance (PELDOR), based on binding affinity.

Main Methods:

• Relaxation induced dipolar modulation enhancement (RIDME) titrations were employed to quantify Cu^II-NTA binding affinities.
• In-cell EPR spectroscopy was performed to monitor Cu^II signal persistence.
• In-cell PELDOR experiments were conducted on sites with differing binding affinities.

Main Results:

• Dissociation constants (K_d) in the 10^-6 and 10^-8 M ranges were determined for β-sheet dHis sites.
• The persistence of the in-cell Cu^II EPR signal directly correlated with binding affinities.
• Analyzable in-cell PELDOR data were obtained only from the high-affinity α-helical site, not the lower-affinity β-sheet sites.

Conclusions:

• Low dissociation constants (K_d) are essential for robust and reliable in-cell distance measurements using endogenous Cu^II-NTA labeling.
• The choice of dHis tag site significantly influences the success of in-cell EPR experiments.
• RIDME provides a sensitive method for characterizing binding affinities relevant to in-cell spin labeling.