Strikingly novel instrumentation for electron paramagnetic resonance (EPR) spectroscopy at the high microwave frequency of 94 GHz (W-band) has been developed. The broad long-term objective of the present proposal is to establish usefulness of this instrument in a significant biomedical application: the study of cholesterol-mediated lipid interactions using dimyristoylphosphatidylcholine (DMPC) membranes containing cholesterol across the phase diagram. Phospholipids, as well as cholesterol, spin-labeled at a number of available sites using either 14N or 15N isotopes will be used. Measurements of bimolecular collision rates that occur between a 14N-tagged site and a 15N-tagged site provide information about the impact of cholesterol on molecular dynamics. The time scale for the experimental methods is on the order of 10 ?s, which is a typical value for the spin-lattice relaxation time, T1, of the spin label. Measurements in the range of 10 times faster to 10 times slower are within reach. This is a range that is considered not only to be of high biological relevance but also to be essentially inaccessible using other instrumental modalities. Two complementary EPR techniques will be used: saturation recovery (SR) and pulse electron-electron double resonance (ELDOR). Excitation in both techniques will be an adiabatic rapid sweep of an intense microwave frequency across a selected region of the EPR spectrum. This is a CHIRP frequency-swept pulse. It is a highly innovative technical approach that requires the use of an arbitrary waveform generator (AWG) and a broadband loop-gap resonator (LGR).
Specific aims are as follows: (1) Development of CHIRP excitation and observation methods for SR followed by measurements across the experimental parameters of the phase diagram using available spin labels. (2) Use of the data of Specific Aim 1 to design analogous ELDOR experiments, which will also be applied across the phase diagram using various spin-label pairs-one with 14N and one with 15N. Measurements of collision frequencies between labels at different depths-so-called "vertical fluctuations"-will be made to provide insight into the mediation of membrane dynamics by cholesterol. The diffusion-in-a-cone model will be tested using 14N/15N pairs, each member of the pair at the same depth, as a function of increasing depth, across the phase diagram. And (3), which is an intense state-of-the-art engineering initiative: direct digital detection of the microwave carrier in the existing instrument. A novel aspect of Aim 3 is the use of magnitude detection of SR and ELDOR signals-which is new in the context of EPR and is particularly appropriate when using CHIRP pulses. This proposal rests on the highly significant hypothesis that molecular structures require knowledge of molecular dynamics to be biologically relevant. Extension of the methodology to membrane-bound proteins is foreseen.

Public Health Relevance

Molecular dynamics occur in all biological molecules across a wide range of motional frequencies, some of which are central to biological function. The nitroxide-radical spin-label method, based on electron paramagnetic resonance (EPR), is suitable for the study of molecular dynamics, including dynamics with characteristic motional periods of a millisecond to a microsecond that are biologically- relevant. The proposal applies a new EPR spectrometer that has many innovative features and operates at the high microwave frequency of 94 GHz to characterize cholesterol-mediated lipid interactions in membranes as a function of cholesterol content and temperature.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB002052-36
Application #
8645625
Study Section
Instrumentation and Systems Development Study Section (ISD)
Program Officer
Sastre, Antonio
Project Start
1979-07-01
Project End
2016-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
36
Fiscal Year
2014
Total Cost
$568,482
Indirect Cost
$189,365
Name
Medical College of Wisconsin
Department
Biophysics
Type
Schools of Medicine
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Widomska, Justyna; Zareba, Mariusz; Subczynski, Witold Karol (2016) Can Xanthophyll-Membrane Interactions Explain Their Selective Presence in the Retina and Brain? Foods 5:
Subczynski, Witold K; Mainali, Laxman; Raguz, Marija et al. (2016) Organization of lipids in fiber-cell plasma membranes of the eye lens. Exp Eye Res :
Zareba, M; Widomska, J; Burke, J M et al. (2016) Nitroxide free radicals protect macular carotenoids against chemical destruction (bleaching) during lipid peroxidation. Free Radic Biol Med 101:446-454
Kittell, Aaron W; Hyde, James S (2015) Spin-label CW microwave power saturation and rapid passage with triangular non-adiabatic rapid sweep (NARS) and adiabatic rapid passage (ARP) EPR spectroscopy. J Magn Reson 255:68-76
Mainali, Laxman; Raguz, Marija; O'Brien, William J et al. (2015) Properties of membranes derived from the total lipids extracted from clear and cataractous lenses of 61-70-year-old human donors. Eur Biophys J 44:91-102
Raguz, Marija; Mainali, Laxman; O'Brien, William J et al. (2015) Lipid domains in intact fiber-cell plasma membranes isolated from cortical and nuclear regions of human eye lenses of donors from different age groups. Exp Eye Res 132:78-90
Raguz, Marija; Mainali, Laxman; O'Brien, William J et al. (2015) Amounts of phospholipids and cholesterol in lipid domains formed in intact lens membranes: Methodology development and its application to studies of porcine lens membranes. Exp Eye Res 140:179-86
Mainali, Laxman; Vasquez-Vivar, Jeannette; Hyde, James S et al. (2015) Spin-labeled small unilamellar vesicles with the T 1-sensitive saturation-recovery EPR display as an oxygen sensitive analyte for measurement of cellular respiration. Appl Magn Reson 46:885-895
Raguz, Marija; Mainali, Laxman; O'Brien, William J et al. (2014) Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens. Exp Eye Res 120:138-51
Mainali, Laxman; Sidabras, Jason W; Camenisch, Theodore G et al. (2014) Spin-label W-band EPR with seven-loop-six-gap resonator: Application to lens membranes derived from eyes of a single donor. Appl Magn Reson 45:1343-1358

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