SLIM: A Short-Linked, Highly Redox-Stable Trityl Label for High-Sensitivity In-Cell EPR Distance Measurements.

Area of Science:

• Biophysical Chemistry
• Molecular Biology
• Biochemistry

Background:

• Understanding biomolecular function requires knowledge of structure and dynamics under native conditions.
• Pulsed dipolar EPR spectroscopy (PDS) with site-directed spin labeling (SDSL) is crucial for biophysical studies.
• Existing spin labels have limitations for in-cell applications, including poor stability and long linkers.

Purpose of the Study:

• To develop a novel spin label for improved in-cell biophysical measurements.
• To overcome the limitations of current spin labels for PDS and SDSL applications.
• To investigate conformational changes of biomolecules within living eukaryotic cells.

Main Methods:

• Synthesis of a novel trityl radical-based spin label (SLIM) with a maleimide-functionalized methylene group.
• Application of SLIM in site-directed spin labeling (SDSL) for pulsed dipolar EPR spectroscopy (PDS).
• In-cell distance measurements and conformational analysis of the GDI domain of Yersinia outer protein O (YopO).

Main Results:

• The developed SLIM label exhibits high radical stability and resistance to reduction.
• SLIM enables sensitive distance measurements down to 90 nm concentrations with narrow distance distributions.
• Conformational changes in the YopO GDI domain were detected within eukaryotic cells using SLIM.

Conclusions:

• The novel SLIM spin label significantly advances in-cell EPR spectroscopy capabilities.
• SLIM provides a robust tool for studying biomolecular structure and dynamics in native cellular environments.
• This work demonstrates the utility of SLIM for real-time conformational analysis of proteins in vivo.