A Two-Armed Probe for In-Cell DEER Measurements on Proteins*

Area of Science:

• Biophysics
• Structural Biology
• Chemical Biology

Background:

• Site-directed spin labeling (SDSL) with double electron-electron resonance (DEER) is crucial for measuring distances in biological systems.
• Existing spin labels face challenges in cellular environments, including reductive dissociation and probe rigidity.
• Developing robust probes is essential for accurate in-cell structural biology.

Purpose of the Study:

• To synthesize and evaluate novel two-armed gadolinium (GdIII) complexes (GdIII-CLaNP13a/b/c) for in-cell DEER measurements.
• To assess the stability and performance of a thioether linkage in resisting cellular reducing conditions.
• To determine inter-spin distances in T4 Lysozyme variants within cellular environments.

Main Methods:

• Synthesis of three novel two-armed GdIII complexes featuring a thioether linkage.
• Site-directed spin labeling of T4 Lysozyme variants (N55C/V57C/K147C/T151C) with the synthesized GdIII probes.
• Distance measurements using 95 GHz DEER in vitro, in cell lysate, and in live Dictyostelium discoideum cells.

Main Results:

• Successful synthesis of GdIII-CLaNP13a/b/c complexes with thioether linkages.
• DEER measurements of doubly labeled T4 Lysozyme yielded distances of 4.5 nm, consistent with paramagnetic NMR data.
• The probes demonstrated complete and durable labeling with narrow distance distributions, even within live cells, indicating probe rigidity.

Conclusions:

• The novel GdIII-CLaNP probes with thioether linkages are stable and effective for in-cell DEER measurements.
• Dual attachment sites contribute to probe rigidity and precise distance determination in complex biological milieu.
• This methodology advances the application of EPR spectroscopy for in-cell structural studies of proteins and protein complexes.