This competitive renewal proposal is to develop pulse saturation-recovery (SR) electron paramagnetic resonance (EPR) for application to spin labels in a context of site directed spin labeling (SDSL). The applicant's laboratory has recently made a breakthrough in SR detection based on a novel digital sub-sampling strategy, which has been termed time locked sub-sampling, and a new pulse spectrometer is under construction. Thorough engineering evaluation and design optimization including signal processing software are proposed for the next funding period. Feasibility for a new X-band bimodal loop gap sample resonator has also been demonstrated and further innovation is proposed. Engineering-based development follows naturally from the work of the previous funding period. SR can measure the rate of bimolecular collisions of spin labels with oxygen, and is a primary methodology for studying oxygen transport. Spin label oximetry is an important tool in identification of structural motifs such as alpha-helices or beta-sheets in proteins through SDSL methods. Three model systems are used here in each of three application-based specific aims. The model systems, in order of increasing biological relevance, are maleimide spin label in super-cooled sec-butylbenzene, hemoglobin labeled at beta93 in glycerol-water mixtures, and the ferric enterobactin receptor, Fep-A. The SR method is validated and compared with more conventional methodology. A novel SR method to separate overlapped spectra, termed discrimination by oxygen transport, is used to characterize multiple motional components of FepA. SR apparatus acquires intense free induction decay (FID) signals that are normally suppressed. Instead, this investigator proposes to use FID signals to measure site-to-site distances in double-labeled FepA. Frequency swept pulse electron-electron double resonance is introduced to characterize slow restricted angle motions in Fep-A in a situation where overall motion of the protein is negligible. The detection method, the bimodal resonator, use of FID signals for distance determination, use of SR to separate overlapped spectra, and measurement for the first time of restricted-angle motions are thought to be highly innovative. SDSL is a new and rapidly expanding methodology in structural biology. Addition of saturation recovery to SDSL methodology will, we hypothesize, revolutionize the field. The proposal lies at the foundation of modern biomedical research: development of tools that enhance one's ability to determine protein structure, dynamics and function. The investigator asserts that only on such a foundation can a rational approach to improved human health be based.