Electron paramagnetic resonance (EPR) spectroscopy can provide information about the structure and dynamics of biomacromolecules in physiologically relevant conditions. Its inherently high sensitivity is still inadequate for some mass-limited samples -- for example, membrane proteins -- which are notoriously difficult to express, purify, and crystallize. This project aims to decrease the limit of detection for inductive-detection EPR spectroscopy at room temperature, so that low-yield biomacromolecular samples can be studied under physiologically relevant conditions. To achieve this objective, we will use a novel resonator design that bridges the gap between planar microresonators and conventional cavity resonators. Our novel planar inverse anapole microresonator design provides nanoliter active volumes combined with high quality factors, providing a projected improvement of two orders of magnitude in sensitivity at room temperature.
Our first aim i s to design and fabricate microresonators for operation at 9 GHz and 34 GHz. First, we will carry out finite element simulations of the field distributions and reflection coefficients for resonators coupled to waveguides. Based on these results, we will optimize the device geometry and dimensions to obtain nanoliter magnetic-field hotspots and high quality-factors. We will fabricate these optimized resonators at the NIST Nanofabrication facility. Next, we will characterize the microresonators and integrate them into a commercial 9 GHz and home-built 34 GHz EPR spectrometer.
Our second aim i s to demonstrate the viability of these resonators for structural biology EPR spectroscopy experiments. To do this, we will first design and fabricate microfluidic devices capable of localizing nanoliter sample volumes in the magnetic hotspot volume of the microresonator. To validate the device performance, we will use a concentration series of spin-labeled peptides. Finally, to demonstrate applicability to a biomacromolecular sample, we will study the Gprotein coupled receptor melanopsin. If successfully implemented, this resonator design will achieve an unprecedented sensitivity for EPR spectroscopy, broadening its applicability to low-yield biomacromolecular samples whose structures are currently poorly understood.
Most current drugs target membrane proteins, with 30-50 % of drugs affecting G-protein coupled receptors. However, the structure-function relationships for this class of proteins are poorly understood due to challenges associated with expression and purification in adequate quantities, hindering rational drug design. This project aims to significantly increase the EPR capabilities of biomedical research laboratories, especially for interrogating mass-limited samples so that structural biology experiments can be conducted with volumes and concentrations two orders of magnitude smaller than currently possible.
