DEER (double electron electron resonance) is a pulsed EPR (electron paramagnetic resonance) spectroscopy technique that is used to measure distances between unpaired electrons. DEER can be applied in structural biology to accurately characterize the structure of spin-labeled proteins, at the scale of several nanometers. EPR is commonly conducted using microwave frequencies at around 10 GHz (X-band), but spectral resolution and sensitivity can be improved by increasing the microwave frequencies to around 35 GHz (Q-band). Microwaves are concentrated on the sample of interest through the use of a resonator. Resonators developed for use with traditional continuous wave (CW) EPR experiments are designed with a high quality factor (i.e. most efficient storage of microwave energy) at a single resonance frequency. However, DEER requires a wide resonance bandwidth, which means that the quality factor of the resonator must be lowered, often at the expense of sensitivity. The goal of this project is to develop a Q-band resonant cavity for use with DEER that combines a bandwidth of 60-70 MHz with high accuracy and sensitivity. The resonator is designed using CST Microwave Studio, an electromagnetics modeling software package, to maximize its bandwidth and power conversion efficiency. A cylindrical cavity geometry is chosen due its ease of construction and simple tuning procedure. We anticipate that, after further development and testing, this initial design will serve as a platform that can be easily modified to accomodate other advanced EPR techniques.

Electron paramagnetic resonance (EPR) spectroscopy is a technique for analyzing chemical species with unpaired electrons (spin centers). An EPR spectrum provides information on the structure of the spin center as well as the total number of spin centers in a sample. The ability to use EPR to quantitatively determine the spin concentration of a sample is contingent on accurate comparison to a known concentration standard. For biological systems, a commonly used standard is a complex of copper(II) and EDTA (ethylenediaminetetraacetic acid); however, the presence of multiple species of copper-ligand complexes results in an EPR spectrum with overlapping signals that are not easily quantifiable. In an effort to develop a more accurate single-species standard, the coordination of EDTA to copper(II) as a function of acidity and its effect on the EPR spectrum is explored. Analysis of these spectra shows a single species of a copper(II) EDTA complex at pH three, with potential for use as a spin concentration standard.

The structure and assembly of a protein determines its function and role in a biological system.  By attaching a pair of spin labels to a protein, it is possible to measure the distance between the two spin labels using double electron-electron resonance spectroscopy (DEER).  A spin label is an organic molecule with an unpaired electron.  The spin label concentration is measured using an electron paramagnetic resonance (EPR) spectrometer.  There is a positive linear relationship between the amplitude of a spectra and the concentration of spin labels.  The goal of this experiment is to develop a protocol that will accurately and reproducibly measure spin label concentration in a sample.  This is achieved by obtaining spectra for 10, 20, 50, 100, and 200 uM of TEMPO spin labels so we can determine a standard calibration curve.  By obtaining more spectra, we will be able to minimize the discrepancies of the amplitude based on the calibration curve for a given concentration.  Using the calibration curve, the number of pairs of spin labels that are attached to a protein in a given protein sample can be determined using DEER.  Establishing this protocol is important because it will allow us to monitor the labeling efficiency which is crucial for the distance measurements.

Many important enzymatic reactions require the transport of electrons to function properly. This transport is commonly achieved by passing an electron to several different residues on the peptide chain which composes the enzyme. Tryptophan, a common amino acid, participates in this electron transfer by way of an oxidized radical tryptophan intermediate. We hypothesize that hydrogen bonding characteristics have significant influence on the electron transfer behavior of tryptophan. Understanding the effects and implications of hydrogen bonding would greatly affect how we engineer enzyme structure for reactions involving this type of mechanism. Using EPR (electron paramagnetic resonance) spectroscopy, we recently discovered that the strength and position of the hydrogen bond correlates directly to shifts in the g-tensor, a proportionality constant used to describe the resonance condition of the radical electron. Using computational chemistry software, we are gathering data to better understand how g-tensors change as the geometry of the hydrogen bond is altered. Our main goal is to determine trends which describe how changing specific structural features alters the corresponding g-tensor values of the radical tryptophan. We hope these trends will allow us to understand if and how hydrogen bonding is occurring at a radical tryptophan.