New High-Sensitivity EPR System Advances Protein Dynamics Research

Scientists in the Department of Biophysics have developed a high-sensitivity, stopped-flow electron paramagnetic resonance (EPR) spectroscopy system capable of resolving millisecond timescale conformational exchange rates in spin-labeled proteins. As discussed in the Protein Science article titled “A High-Sensitivity Stopped-Flow EPR System to Monitor Millisecond Conformational Kinetics in Spin-Labeled Proteins,” this EPR system represents a significant step forward in stopped-flow EPR technology used to study protein dynamics.

Proteins are inherently dynamic molecules whose functional motions, from loop rearrangements to secondary structure shifts, play pivotal roles in biological processes such as catalysis, signaling, and molecular interactions. These conformational changes, often occurring on microsecond or slower timescales, are governed by an energy landscape that dictates the populations and interconversion rates of these discrete states. The time dependence of biomolecular structural changes remains underexplored yet is essential for defining the roles of transient states and dynamically driven allostery in protein function.

The newly developed EPR system enables investigation of protein conformational dynamics on the millisecond timescale while dramatically reducing sample requirements compared with existing instruments. This is accomplished through the incorporation of several key innovations: a custom dielectric resonator, optimized sample cell geometry, and an integrated stopped-flow mixer assembly that works seamlessly with an EPR spectrometer.

This system maintains exceptional detection sensitivity while minimizing sample waste, making it possible to conduct time-resolved studies on complex, biologically relevant proteins that are often only available in limited quantities. The article presents two initial applications to showcase this advancement, an analysis of T4 lysozyme folding pathways through site-specific kinetic measurements and the detection of time-resolved allosteric conformational changes in a complex membrane protein system (β2-adrenergic receptor). This technology expands the scope of stopped-flow EPR applications, opening new avenues for investigating dynamic mechanisms in biomedically critical processes that were previously inaccessible due to sample limitations.

Authors of the study are Alexander M. Garces, Richard R. Mett, PhD, Candice S. Klug, PhD, Jason W. Sidabras, PhD, and Michael T. Lerch, PhD. This work was supported by the National Institute of General Medical Sciences.