#1 Frequency Modulation ST EPR Studies of Macromolecular Dynamics at W-band. Albert H. Beth, Department of Molecular Physiology and Biophysics, Vanderbilt University.
The overall aims of this collaborative study are to determine the advantages of microwave frequency modulation for saturation transfer EPR (ST-EPR) studies of macromolecular dynamics at W-band. This project will combine the expertise of the National Biomedical ESR Center in design and construction of this novel new technology with the long-standing interest of the Beth laboratory in studies of very slow rotational dynamics of proteins and their macromolecular assemblies including advanced analyses of experimental data in terms of anisotropic rotational diffusion and constrained rotational diffusion. W-band is predicted to be superior to lower frequencies (e.g., X-band)^ for these demanding applications. The specific aims that will be addressed are:
(1) to document the sensitivity of fm-ST-EPR at W-band to the global isotropic rotational diffusion of a well characterized model protein in solution as a function of modulation frequency; and (2) to extend this work to detailed characterization of the constrained anisotropic rotational diffusion of a transmembrane protein, the anion exchange protein, in the human erythrocyte membrane.
#2 Applications of Information Geometry to the Analysis of NARS Spectroscopy. Keith A. Earle, Department of Physics, University of Albany (SUNY).
The specific objective of this collaboration is to use advanced statistical techniques and recent results from communication theory in order to fully characterize spin-spin interactions in the presence of coherent noise over a wide variety of conditions. This collaboration has direct applications for optimizing experimental parameters and in the analysis of dipolar distances up to 40A using the instrumental capability being further developed in TR&D Project 2.
Collaborative Project 2. Specific Aims
Specific Aim 1.1. Survey the parameter space and characterize an informative signal representation of non-adiabatic rapid sweep (NARS) experiments for use in determining interspin distances upwards of 40 A.
Specific Aim 1.2. Assess the effects of various signal-processing filter techniques using an information metric as a way to quantify the influence of filtering on the parameter estimation problem.
#3 Native Structure of Potassium Channels. Adrian Gross, Rosalind Franklin University of Medicine and Science, North Chicago, IL
We intend to measure distances between nitroxide spin labels at room temperature in the 20-30 A range.
Two types of potassium channel samples will be studied: (1) Single mutants where the interspin distance is generated by the symmetry of the channel, and (2) double mutants where the interspin distance corresponds to a non-symmetry-related spin pair. Both sample types are routinely generated and studied in the laboratory.
#4 High Pressure Q-Band NARS. Wayne L. Hubbell, Department of Ophthalmology and Chemistry and Biochemistry, University of California, Los Angeles
The aim of this project is to determine structural constraints on low-lying excited (“invisible”) states of spin-labeled proteins populated by pressure.
#5 Distance Measurements by L-Band NARS. Eric J. Hustedt, Department of Molecular Physiology and Biophysics, Vanderbilt University
The specific objectives of this project center on the use of the L-band NARS technology®® recently developed at the National Biomedical EPR Center at MCW to measure long range (>20 A) distance distributions in spin-labeled protein samples. The overall goal is to develop an understanding of how temperature and solvent conditions affect the measured distance distributions. It is anticipated that this work will have a major impact on our knowledge of how commonly used experimental conditions influence the results obtained.
Specific Aim 1. Using either T4L or the CDB3 dimer, compare distance distribution measurements at cryogenic temperatures using DEER with those obtained at much higher temperatures using L-band NARS.
Experiments will be designed to test how temperature effects the distance distributions obtained by changing the distributions of spin-label side-chain rotamers and protein conformers.
Specific Aim 2. Using either T4L or the CDB3 dimer, investigate how different cryoprotectants and viscosity enhancing agents influence the distance distribution measurements made by L-band NARS.
#6 Intermediate Distance Determinations in the Lipid A Transporter MsbA. Candice S. Klug and James S. Hyde, Department of Biophysics, Medical College of Wisconsin
The specific objective of this collaboration is to obtain spin-spin distance information at physiologically relevant temperatures (i.e., noncryogenic) in a range not possible by other instrumental techniques (18-28 A) using a novel approach to an instrumental capability (L-band) being further developed in TR&D Project 2.
Specific Aim 1. Compare the structural organization of MsbA A270T at its permissive and restrictive temperatures using L-band EPR distance determination.
Specific Aim 2. Quantitate the population of the MsbA A270T homodimer in the closed and open conformations.
#7 Role of RPE Melanosomes in Oxidative Stress in Vitro: Effects of Age-Related Modifications on Antioxidant and Pro-Oxidant Properties of the Pigment Granules. Tadeusz Sarna, Department of Biophysics, Jagiellonian University, Krakow, Poland
The main goal of this project is to investigate physicochemical properties of human retinal pigment epithelium (RPE) melanosomes with aging using melanin free radical centers as intrinsic molecular probes of this important biological pigment and the proposed new approach to high frequency EPR spectroscopy. It is expected that this novel application of the segmental microwave frequency sweep from non-adiabatic to adiabatic FID and spin echo Fourier transform EPR at W-band to characterize motional and spectroscopic properties of melanin will provide us with unique information about key chemical and physical changes of the pigment granules induced in the human RPE by aging, which may reduce their protective capacity.
Specific Aim 1. Verify the postulate that aging is accompanied by oxidative modifications of human RPE melanosomes that change spectroscopic parameters of the melanin radicals in a consistent way, pure absorption and dispersion spectra of RPE pigment granules from single donors of different age and of experimentally photoaged porcine RPE melanosomes will be obtained, and selected spectral features of the
EPR signals will be employed to characterize early and late age-related changes of the pigment granules.
Specific Aim 2. Test the hypothesis that chemical changes of RPE melanosomes occurring with aging
differentially affect different parts and constituents of the pigment granules. D-band EPR spectra of
melanosomes from donors of different age and from photobleached porcine RPE will be used in an attempt to spectrally resolve melanin free radical centers that exhibit significant overlap of their EPR spectra at W-band.
Specific Aim 3. Test the hypothesis that aging of human RPE melanosomes and photoaging of porcine pigment granules modify the accessibility of melanin free radical centers to molecular oxygen, solvent molecules and various chemical agents affecting their reactivity. We will apply the developed method of segmental adiabatic sweeps of the microwave frequency at W-band to probe the distribution of phase memory times across the spectrum of melanin radicals in RPE melanosomes from donors of different age and in photobleached porcine melansomes.
Specific Aim 4. Test the hypothesis that age-related changes of RPE melanosomes affect their morphology and nano-mechanical properties such as rigidity and porosity that could alter antioxidant and photoprotective properties of the granules. We will carry out passage experiments at the interface between the ARP and non-adiabatic conditions to measure very slow rotational diffusion of melanin radicals in RPE melansomes from donors of different ages and in partially photobleached porcine RPE pigment granules.
#8 Structural Basis for SNARE-Mediate Membrane Fusion. Yeon-Kyun Shin, Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University
There is compelling evidence that the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) TMDs play an essential role in promoting the fusion pore opening. We hypothesize that the interaction between target membrane (t-) and vesicle associated (v)-SNARE TMDs drives the fusion pore opening. Alternatively, it is possible that the heterodimeric TMD is shaped in such a way that it could stabilize the fusion pore.
Specific Aim 1. Investigate the heterotypic interaction between SNARE transmembrane domains (TMDs) using site-directed spin-labeling (SDSL) EPR. We will determine the structure and the membrane topology of V- and t-SNARE TMD heterodimer using SDSL EPR spectroscopy to investigate the structural basis effusion pore opening.
#9 Enhanced Discrimination of Cholesterol Bilayer Domains in Model and Biological Membranes by Modification of the Electrostatic Surface Potential of the Membrane. Witold K. Subczynski, Department of Biophysics, Medical College of Wisconsin
The specific objective of this collaboration is to develop a new EPR spin-labeling approach for investigation of phase-separated domains in lipid bilayers. Specifically, we will characterize the formation of cholesterol bilayer domains (CBD) in model membranes and in intact fiber cell plasma membranes of the human eye lens.
A new instrumental capability at L-band, which is being further developed in TR&D Projects 1 and 2 (namely, the extremely narrow central line of EPR spectra of spin labels as well as a short spin-lattice relaxation time), will allow increased sensitivity for the discrimination of domains at a higher rate of lipid exchange. These high exchange rates "mix" results from coexisting domains.
Specific Aim 1. Compare effects of water-soluble relaxation agents, neutral NiEDDA and negatively charged CrOx, on EPR spectra (linewidth, line intensity, and spin-lattice relaxation time) of CSL and T-PC in
POPC/POPS membranes as a function of membrane surface potential.
Specific Aim 2. Use optimized conditions as described in Aim 1 to investigate the formation of cholesterol bilayer domains (CBD) and evaluate the cholesterol solubility limit in POPC/POPS membranes.
Specific Aim 3. Use approaches developed in Specific Aims 1 and 2 to determine the cholesterol concentration at which the CBD is formed in membranes made of from the major phospholipids of the lens fiber cell plasma membrane.
#10 In Vivo Surface Chemistry with Applications in Measuring Ionizing Radiation Dose in Human Finger and Toenails. Steven G. Swarts, University of Florida-Gainesville
The specific objective of this collaboration is to advance in vivo oximetry measurements in a subcutaneous tumor model in rats using a non-adiabatic rapid sweep (NARS) spectroscopy detection scheme. This collaboration utilizes the Center's previous research of NARS bridges and the proposed TR&D Project 1 to translate NARS technology to Dartmouth's established in vivo L-band (1.2 GHz) oximetry station.
Specific Aim 1.Optimize instrumentation and data collection parameters for in vivo NARS oximetry.
Specific Aim 2. Apply and assess in vivo NARS oximetry in a subcutaneous tumor model in rats with implantable biocompatible oxygen reporters and resonators.