CW and pulsed Electron Paramagnetic Resonance (EPR) spectroscopic techniques coupled with site-directed spin-labeling (SDSL) can provide important structural information on complicated biological systems such as membrane proteins. Strategically placed spin-labels alter relaxation times of NMR active nuclei and yield pertinent structural information. EPR techniques such as Double Electron-Electron Resonance (DEER) and Electron Spin Echo Envelope Modulation (ESEEM) are powerful structural biology tools. The DEER technique can be used to measure distances between 2 spin labels from 20 to 70 Å. However, the application of DEER spectroscopy to study membrane proteins can be difficult due to short phase memory times (T_m) and weak DEER modulation in more biologically relevant proteoliposomes when compared to water soluble proteins or membrane proteins in detergent micelles. The combination of these factors often leads to broad distance distributions, poor signal to noise, and limitations in the determination of longer distances. The short phase memory times are typically due to uneven distributions of spin-labeled protein within the lipid bilayer, which creates local inhomogeneous pockets of high spin concentrations. Approaches to overcome these limitations and improve the quality of DEER measurements for membrane proteins will be discussed: lipodisq nanoparticles, bi-functional spin labels (BSL), and Q-band pulsed EPR spectroscopy. ESEEM data will be shown to probe the secondary structure of membrane proteins. CW-EPR spectra of spin-labeled membrane proteins will be used to investigate dynamics and the immersion depth in a lipid bilayer.

We are exploring a new methodology to considerably enhance the efficacy of X-ray therapy and diagnostics. Conventional medical X-ray sources for radiation therapy emit X-rays on a broadband bremsstrahlung spectrum, over a wide range of energies. Lower energy X-rays from these sources are absorbed by the skin potentially causing burns. The highest energy X-rays have very low interaction rates with tissue and so require high intensities at the tumor to have any effect. Several types of X-ray sources exist that overcome this energy-inefficiency. Synchrotrons are capable of producing tunable monochromatic, single-energy X-rays; however, they are biomedically impractical due to massive size and cost. New methods of generating X-rays have recently seen a surge in development. Quasi-monochromatic X-ray sources are newer and produced with a variety of methods including high intensity lasers present on many university campuses or filtering techniques. These sources are approaching monochromaticity and are more practical in size and cost compared to synchrotrons. Monochromatic and quasi-monochromatic X-rays are advantageous over broadband sources for several reasons. They can be tuned more directly to deep-seated tumors and to target specific energies in heavy element nanoparticles embedded in tumors to release localized cascades of high energy photons and electrons to more efficiently kill cancer cells while leaving healthy cells intact. We propose a study of monochromatic and quasi-monochromatic X-ray devices to better understand how their spectra interact with cells, tissue, and heavy atoms for enhancing biomedical therapy in order to reduce the amount of normal, healthy tissue that is irradiated.

Water dynamics on the protein surface mediate both protein structure and function. However, there remain many questions about the role of the protein hydration layer in protein fluctuations and how the dynamics of this layer directly relates to specific protein properties. The fish eye lens protein γM7-Crystallin are found in extremely high concentrations in vivo at concentrations nearing the packing limit, higher than 1000 mg/ml, corresponding to only a few water layers between proteins. In this study, we conducted a site-specific probing of the γM7-Crystallin hydration layer and side chain dynamics at 9 sites around the surface using a tryptophan scan with femtosecond spectroscopy. We observed three types of hydration-water relaxations and two types of protein sidechain motions which fluctuate on the picosecond timescale. These motions are heterogeneous over the protein surface and correlate to the various steric and chemical properties of the local protein environment.