Biology subjects

Mett, R. R.

Publications and source records attributed to Mett, R. R..

3 recordsLinked to original sources

bioRxiv2025

Improved EPR sensitivity for aqueous biological samples using low-volume multi-channel cells and dielectric resonators

Reducing sample volumes for electron paramagnetic resonance (EPR) spectroscopy applications places increasing demands on hardware design to preserve or enhance EPR signal intensity. In this work, the design, fabrication, and testing of dielectric resonators and multi-channel aqueous sample cells for applications in X-band (nominally 9.5 GHz) EPR is presented. Our aim was to maximize the EPR signal intensity for sample sizes of 3-4 L and 200 nL. These advances are summarized as follows: single-crystal sapphire and rutile dielectric resonators with very low loss tangent and high resonator efficiency; minimum dielectric resonator coupling to radiation shield to reduce ohmic losses; 3D printed aqueous sample cells with thin multi-channel construction to minimize radio-frequency dissipation in the sample; and a Gordon coupler for maximum coupling range and minimum stored energy to eliminate frequency shifts during tuning. Sample cell geometries were designed by leveraging insights gained from analytic theory to inform finite-element modeling of electromagnetic fields. Experimental comparisons of multi-channel sample cells using a sapphire resonator exhibited a 2.2-fold increase in EPR signal intensity compared with a standard capillary at 3-4 L, while simulations predict an additional 23% improvement with further 3D printing advances. For samples at 200 nL, a rutile dielectric resonator with a multi-channel sample cell was simulated to improve EPR sensitivity by a 2.7-fold increase compared with a capillary at the same volume.

biophysics↗

bioRxiv2025

A High-Sensitivity Stopped-Flow EPR System to Monitor Millisecond Conformational Kinetics in Spin-Labeled Proteins

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 {beta}2 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.

biophysics↗

bioRxiv2024

A pressure-jump EPR system to monitor millisecond conformational exchange rates of spin-labeled proteins

Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) using nitroxide spin labels is a well-established technology for mapping site-specific secondary and tertiary structure and for monitoring conformational changes in proteins of any degree of complexity, including membrane proteins, with high sensitivity. SDSL-EPR also provides information on protein dynamics in the time scale of ps-s using continuous wave lineshape analysis and spin lattice relaxation time methods. However, the functionally important time domain of s-ms, corresponding to large-scale protein motions, is inaccessible to those methods. To extend SDSL-EPR to the longer time domain, the perturbation method of pressure-jump relaxation is implemented. Here, we describe a complete high-pressure EPR system at Q-band for both static pressure and millisecond-timescale pressure-jump measurements on spin-labeled proteins. The instrument enables pressure jumps both up and down from any holding pressure, ranging from atmospheric pressure to the maximum pressure capacity of the system components ([~]3500 bar). To demonstrate the utility of the system, we characterize a local folding-unfolding equilibrium of T4 lysozyme. The results illustrate the ability of the system to measure thermodynamic and kinetic parameters of protein conformational exchange on the millisecond timescale.

biophysics↗