This is an instrumentation development proposal for improved electron paramagnetic resonance (EPR) spectroscopy for biomedical research. Specifically, the applicants propose to develop saturation recovery (SR) EPR and SR electron-electron double resonance (ELDOR) apparatus at high microwave frequencies that is tailored for experiments using nitroxide radical spin labels and spin probes, in the context of site-directed spin labeling (SDSL). These studies help to determine the dynamics of molecular structure. SR is often used to study bimolecular collisions of oxygen with spin labels. Measurement of oxygen accessibility in SDSL permits identification of structural motifs of proteins. In this proposal, focus not only is on oximetry but also on the use of SR EPR and SR ELDOR to study slow rotational diffusion of proteins. This laboratory pioneered extension of SR instrumentation to high microwave frequencies. The first SR instrument at Q-band (35 GHz) was developed in the previous funding period, and the first at W-band (94 GHz) in the current funding period. It was discovered that the loop-gap resonator (LGR) yields outstanding performance because of high bandwidth and high efficiency in concentrating the microwave field intensity.
In Aim 1 of this proposal, extension to D-band (144 GHz) using an LGR is proposed. The features of the relatively immobilized spin-labeled protein no longer overlap at this frequency, which is highly advantageous. The design is based on an innovative combination of microwave frequency translation and low order multiplication.
In Aims 2 and 3, SR technology at W-band (94 GHz) will be enhanced. As in Aim 1, these two aims focus on application to proteins undergoing slow rotational diffusion.
In Aim 2, the level of microwave power will increase, the pulses will become shorter, and the dead-time will be decreased: all of which will improve SR EPR and SR ELDOR in the study of slow rotational diffusion. The technique of inversion recovery (IR) will be developed, which in comparison with SR will improve the SNR by two times and improve temporal resolution by about ten times.
In Aim 3, another way to excite the spin system is introduced: use of adiabatic rapid passage (ADR) by sweep of the microwave frequency from one point in the spectrum to another to excite the spins. The discovery that LGRs are useful at high microwave frequencies because of high bandwidths is central to Aim 3. A state-of-the-art arbitrary waveform generator (AWG) is an enabling technology for frequency swept ADR excitation.
Aims 2 and 3 are expected to be complementary, with the methods of Aim 2 being on a shorter timescale and the methods of Aim 3 providing more uniform excitation over well-defined regions of the spectrum. The engineering team is very experienced and is considered to be a national resource. This proposal contributes to the nation's infrastructure for biomedical research. Developed instruments become available to all researchers through the National Biomedical EPR Center, a P41 Research Resource directed by the PI. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
2R01EB002052-30
Application #
7524191
Study Section
Instrumentation and Systems Development Study Section (ISD)
Program Officer
Mclaughlin, Alan Charles
Project Start
1979-07-01
Project End
2012-03-31
Budget Start
2008-06-01
Budget End
2009-03-31
Support Year
30
Fiscal Year
2008
Total Cost
$586,681
Indirect Cost
Name
Medical College of Wisconsin
Department
Biophysics
Type
Schools of Medicine
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Subczynski, Witold Karol; Widomska, Justyna; Mainali, Laxman (2017) Factors Determining the Oxygen Permeability of Biological Membranes: Oxygen Transport Across Eye Lens Fiber-Cell Plasma Membranes. Adv Exp Med Biol 977:27-34
Sidabras, Jason W; Sarna, Tadeusz; Mett, Richard R et al. (2017) Uniform field loop-gap resonator and rectangular TEU02 for aqueous sample EPR at 94GHz. J Magn Reson 282:129-135
Subczynski, Witold K; Mainali, Laxman; Raguz, Marija et al. (2017) Organization of lipids in fiber-cell plasma membranes of the eye lens. Exp Eye Res 156:79-86
Strangeway, Robert A; Hyde, James S; Camenisch, Theodore G et al. (2017) Broadband W-band Rapid Frequency Sweep Considerations for Fourier Transform EPR. Cell Biochem Biophys 75:259-273
Sidabras, Jason W; Mett, Richard R; Hyde, James S (2017) Extruded dielectric sample tubes of complex cross section for EPR signal enhancement of aqueous samples. J Magn Reson 277:45-51
Widomska, Justyna; Zareba, Mariusz; Subczynski, Witold Karol (2016) Can Xanthophyll-Membrane Interactions Explain Their Selective Presence in the Retina and Brain? Foods 5:
Widomska, Justyna; Zareba, Mariusz; Subczynski, Witold Karol (2016) Can Xanthophyll-Membrane Interactions Explain Their Selective Presence in the Retina and Brain? Foods 5:
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
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) 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 140:179-186

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