Developing Novel Radical Protein Footprinting Methods for Application in Living Systems

Author

Lyle TobinFollow

Date of Award

Spring 5-9-2025

Document Type

Undergraduate Thesis

Department

Chemistry and Biochemistry

First Advisor

Joshua S. Sharp

Second Advisor

Eden Tanner

Third Advisor

Saumen Chakraborty

Relational Format

Dissertation/Thesis

Abstract

Radical protein footprinting (RPF) is a family of structural biology techniques that generate radicals in situ to label solvent-accessible amino acid residues. Coupled with liquid chromatography/mass spectrometry, RPF enables the characterization of protein higher order structure and sites of protein-protein and protein-ligand interactions. While traditionally performed in vitro, recent advancements have extended RPF to ex vivo and in vivo applications, including intact cells, nematodes, and 3D cell cultures. RPF in living systems allows the structural analysis of proteins and protein complexes in their native environment, preserving physiological interactions, dynamic conformational changes, and structural contexts that might otherwise be lost or altered in vitro. Beyond basic science, RPF in living systems could identify structural biomarkers, mechanisms of protein therapeutic efficacy, and novel drug targets, among myriad applications. However, despite recent advances, technical barriers have precluded the application of RPF to many biologically and clinically relevant living systems.

This study reviews the history of RPF in living systems and reports the development of novel RPF techniques for both mammalian whole blood and intracellular compartments in bacteria. The sulfate radical anion (SO₄⁻•) precursor sodium persulfate (Na₂S₂O₈), poorly characterized for use in protein footprinting, is shown to be stable in both whole blood and Escherichia coli culture and suspension. The first labeling of both peptides and proteins with SO₄⁻• generated by a UV lamp was performed. A quench and dosimetric technique for SO₄⁻• were developed and validated, and the SO₄⁻• protein footprinting technique was applied to both mammalian whole blood and E. coli. Additionally, the morphological effect of Na₂S₂O₈ on erythrocytes was evaluated to ensure that RPF is performed under native or near-native physiological conditions.

Recommended Citation

Tobin, Lyle, "Developing Novel Radical Protein Footprinting Methods for Application in Living Systems" (2025). Honors Theses. 3287.
https://egrove.olemiss.edu/hon_thesis/3287

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