A high-sensitivity stopped-flow EPR system to monitor millisecond conformational kinetics in spin-labeled proteins. Protein Sci 2025 Aug;34(8):e70214
Date
07/17/2025Pubmed ID
40671309Pubmed Central ID
PMC12267652DOI
10.1002/pro.70214Scopus ID
2-s2.0-105011365823 (requires institutional sign-in at Scopus site)Abstract
Electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for studying biological systems, with applications in drug discovery, protein dynamics, membrane biology, and enzyme mechanisms. However, sample volume requirements and sensitivity limitations have historically constrained time-resolved measurements of protein dynamics using stopped-flow (SF) EPR spectroscopy. To address these challenges, we developed a high-sensitivity SF EPR system featuring a custom dielectric resonator, an optimized low-volume sample tube geometry design, and the SF mixer assembly integrated into the resonator housing. This system significantly reduces sample requirements for the investigation of protein conformational dynamics on the millisecond timescale. We demonstrate its capabilities through two applications: the analysis of T4 lysozyme unfolding kinetics, which revealed site-specific variations in the folding pathway, and the measurement of ligand-induced conformational changes in the β2 adrenergic receptor, a challenging membrane-protein system. This advancement broadens the applicability of SF EPR to complex, biomedically relevant proteins, facilitating studies of protein-protein and protein-ligand interactions in diverse biological processes.
Author List
Garces AM, Mett RR, Klug CS, Sidabras JW, Lerch MTAuthors
Alexander M. Garces Postdoctoral Researcher in the Biophysics department at Medical College of WisconsinCandice S. Klug PhD Professor in the Biophysics department at Medical College of Wisconsin
Michael Lerch PhD Associate Professor in the Biophysics department at Medical College of Wisconsin
Jason W. Sidabras PhD Assistant Professor in the Biophysics department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
Bacteriophage T4Electron Spin Resonance Spectroscopy
Kinetics
Muramidase
Protein Conformation
Receptors, Adrenergic, beta-2
Spin Labels
