Biology subjects

Sidabras, J. W.

Publications and source records attributed to Sidabras, J. W..

5 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

An Order of Magnitude Signal-to-Noise Improvement of Magnetic Resonance Spectra using a Segmented-Overlap Fourier-Filtering and Averaging (SOFFA) Approach.

Segmented-Overlap Fourier-Filtering and Averaging (SOFFA) data acquisition method is described in detail for magnetic resonance spectroscopy. In this work the four processes that encompass the SOFFA data acquisition method are detailed: (i) oversampling spectral segments, (ii) Fourier block-filtering, (iii) segment-overlap averaging, and (iv) decimation. Three experimental examples are shown. Conventional Continuous Wave (CW) Electron Paramagnetic Resonance (EPR) is compared to SOFFA-CW of a single reduced [4Fe-4S]+ (S=1/2) at concentrations of 1 mM, 100 {micro}M, and 10 {micro}M showing an average increase in concentration sensitivity by a factor of 5.6. Experimental comparison of CW and SOFFA nonadiabatic rapid scan (SOFFA-NARS) data with similar filter parameters and field-modulation amplitude demonstrates a factor of 10.3 in signal-to-noise improvement for a 150 {micro}M sitedirected spin-labeled Hemoglobin in 82% glycerol at 18C. Conventional and SOFFA rapid scan (SOFFA-RS) data was collected for a minuscule amount of lithium phthalocyanine (LiPC) and a factor of 9.6 in signal-to-noise improvement is demonstrated. The signal-tonoise improvements were made for the same data acquisition times on standard commercial instruments. This method can be implemented to perform real-time segmented processing and, combined with more sophisticated averaging methods, will push the state-of-the-art sensitivity in magnetic resonance spectroscopy.

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

Asymmetric spin echo multi-echo echo planar imaging (ASEME-EPI) sequence for pre-clinical high-field fMRI

In functional magnetic resonance imaging (fMRI) of the blood oxygen level-dependent (BOLD) contrast, gradient-recalled echo (GRE) acquisitions offer high sensitivity but suffer from susceptibility-induced signal loss and lack specificity to microvasculature. In contrast, spin echo (SE) acquisitions provide improved specificity at the cost of reduced sensitivity. This study introduces Asymmetric Spin Echo Multi-Echo Echo Planar Imaging (ASEME-EPI), a technique designed to combine the benefits of both GRE and SE for high-field preclinical fMRI. ASEME-EPI employs a spin echo readout followed by two asymmetric spin echo (ASE) GRE readouts, providing an initial T2-weighted SE image and subsequent T2*-weighted ASE images. A feasibility study for the technique was implemented on a 9.4 T pre-clinical MRI system and tested using a visual stimulation in northern tree shrews. Comparing ASEME-EPI with conventional GRE echo planar imaging (GRE-EPI) and SE echo planar imaging (SE-EPI) acquisitions, results showed that ASEME-EPI achieved BOLD contrast-to-noise ratio (CNR) comparable to GRE-EPI while offering improved specificity in activation maps. ASEME-EPI activation was more confined to the primary visual cortex (V1), unlike GRE-EPI which showed activation extending beyond anatomical boundaries. Additionally, ASEME-EPI demonstrated the ability to recover signal in areas of severe field inhomogeneity where GRE-EPI suffered from signal loss. The performance of ASEME-EPI is attributed to its multi-echo nature, allowing for SNR-optimized combination of echoes, effectively denoising the data. The inclusion of the initial SE also contributes to signal recovery in areas prone to susceptibility artifacts. This feasibility study demonstrates the potential of ASEME-EPI for high-field pre-clinical fMRI, offering a promising compromise between GRE sensitivity and SE specificity while addressing challenges of T2* decay at high field strengths.

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↗