PROJECT SUMMARY/ABSTRACT: Electron paramagnetic resonance (EPR) is a spectroscopic technique that measures the absorption of energy by unpaired electrons and is used to monitor interactions with the local molecular environment. These unpaired electrons can naturally occur during the catalytic process of an enzyme or be engineered using site-directed spin labeling. Studying these paramagnetic states in detail is critical for understanding the protein structure–function relationship of the unpaired electrons to coordination sphere and secondary structures of the protein. In this proposal, I focus on three technical and method developments at X- band (nominally 9.5 GHz) that will significantly improve EPR spectroscopy for the biomedical research community. I will (i) enhance the EPR sensitivity of the self-resonant microhelix for protein single-crystal EPR of small to medium-sized (0.1–3 nl) crystals, (ii) establish true free induction decay detected EPR for volume-limited frozen samples (85 nl), and (iii) develop a new resonator, the self-resonant microspiral, for advanced time- domain continuous-wave (CW) experiments with microfluidic (500 nl) sample handling. First, enhancements to a key enabling technology, the self-resonant microhelix, will improve EPR sensitivity by an order of magnitude due to application of an innovative matching circuit and cryogenic low-noise amplifier. To improve adoption of this prototype, I will implement a more standard workflow for protein crystal handling, including a computer- controlled goniometer. The prototype will be designed to easily integrate into a commercial X-band pulse spectrometer. Because the self-resonant microhelix has a measured resonator efficiency parameter of 3.2 mT/W1/2, which is greater than 5 times that of commercially available resonators, the power required for a typical 80 ns pulse is reduced by 3 orders of magnitude (from 45 W to just 43 mW). Reduced incident power and implementation of an innovative 3-port transmission line coupling scheme with an onboard cryogenic low-noise amplifier will establish a resonator deadtime less than 5 ns. By leveraging these characteristics, I can develop a new spectrometer prototype for true free induction decay detected EPR, which will drastically improve the EPR signal intensity of biological samples and allow for advanced pulse methodologies that are currently not possible with commercial X-band EPR spectrometer design. Finally, I will introduce a new micro-resonator, the self- resonant microspiral, which will increase the concentration sensitivity for CW EPR by a factor of 70 compared with the microhelix, transforming microfluidic EPR into a viable tool for drug discovery. The self-resonant microspiral enables a new incipient adiabatic passage experiment, pioneered here, that monitors changes of T1 and T2 with changes of the microenvironment of the unpaired electron. This experiment is supported by new sample acquisition methodology that increases CW and adiabatic rapid scan sensitivity by an order of magnitude for the same measurement time. In total, these key enabling technologies will further the adoption of nano-EPR, where performing EPR experiments on volume-limited samples less than 500 nl at X-band becomes practical.

PROJECT NARRATIVE Electron paramagnetic resonance (EPR) spectroscopy is a critically important technique in biomedical research with a unique ability to detect naturally occurring or engineered unpaired electrons in complex biological environments. I focus on the development of three technical and method developments that significantly improve X-band EPR sensitivity for three classes of samples: small to medium-sized (0.1–3 nl) protein single crystals, volume-limited frozen samples (85 nl), and microfluidic (500 nl) with microfluidic sample handling. The combination of these prototypes will be easy to use and widely available to the scientific community, enabling a wide range of new applications in biomedical EPR spectroscopy.