An electron paramagnetic resonance (EPR) saturation recovery (SR) and pulse electron-electron double resonance (ELDOR) capability will be developed at W-band (94 GHz) that is tailored for application to nitroxide spin labeled biomolecules in the aqueous phase. This capability does not presently exist in any laboratory. Most pulse experiments at X-band (9.5 GHz) are carried out using loop gap resonators (LGR), which have the dual advantages of low Q-value, thereby increasing the bandwidth, and high sensitivity for recombinant protein samples of limited availability, as occurs in site-directed spin-labeling (SDSL) experiments. One aspect of this proposal is the exciting discovery of a cavity resonator at W-band that has a similar bandwidth, uses a similar amount of material, and will result in more than 10 times increase in sensitivity compared with an LGR at X-band.
In Aim 1, we propose to develop three novel W-band resonators, two of which are Uniform Field (UF) cavity resonators that were introduced during the previous funding period at X-band.
In Aim 2, we propose to develop a multi-arm frequency-translation accessory that translates the output of the pulse Q-band bridge (also developed during the previous funding period) to W-band for sample irradiation and back to Q-band for digital detection. This cost-effective strategy also provides efficient use of scarce engineering resources. The proposed automatic frequency control (AFC) is novel.
In Aim 3, we will use W-band pulse methods to address two significant spin-label applications. First, we propose to carry the method of Discrimination by Oxygen Transport (DOT) to a new level in two ways: measurement of the anisotropy of oxygen transport in membranes and use of the stretched exponential to characterize distributions of bimolecular collisions between oxygen and spin labels. The second application is resolution of two-component spectra in SDSL using both T1 and oxygen-accessibility differences. The rationale for both depends on the approach to the spectral rigid limit that occurs as the microwave frequency increases and on the longer T1 values that are predicted at W-band by extrapolation from Q-band experiments during the previous funding period. Pulse methodology development at W-band for spin-label studies is timely. Information on protein and membrane dynamics can be obtained on a time scale that is believed not only to be inaccessible using other modalities, but also to be of high biological relevance.
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