This proposal is for continuation of funding of the National Biomedical Electron Paramagnetic Resonance (EPR) Research Resource for years 27 through 31. This Research Resource is broadly based, with unique instrumentation in many branches of EPR spectroscopy. Eight faculty members with EPR training enhance collaborations. Technological Research and Development (TR & D) contains two Sections: Device Design Driven and Methodological Development. In the first, it is proposed to purchase a Bruker W-Band accessory to an ELEXSYS EPR spectrometer and to enhance the high frequency capability by improved frequency translation technology, which will permit use of advanced EPR methods, including time-locked sub-sampling (TLSS) detection and multiquantum EPR at high field strength. Enhancement of W-band capabilities in the liquid phase is also proposed. In addition, three new categories of resonators were discovered in the previous funding period, and it is proposed to develop practical EPR structures based on these discoveries. The second Section of TR&D contains four sub-sections: Methodology Development at W-band, Site- Directed Spin Labeling using Saturation Recovery as well as W-band, Spin Trapping Using Loop Gap Resonators at X- and Q-band, and Measurement of Rapid Reactions Using Loop Gap Resonators with the ELEXSYS X-band Spectrometer. Seven specific Collaborations are described, each written by a different EPR Center faculty member, together with one of their colleagues from another institution or department. The existing young investigator Training program will be continued. Postdoctoral fellows will be appointed for enhanced Training at the EPR Center. A workshop on advanced EPR instrumentation will be held as part of the Dissemination activities of the Center. A special Service project is proposed: development of a YIG oscillator to replace klystrons. EPR spectroscopy is used to study biomolecular structure and function, including spin-labeled macromolecules, metalloproteins, and free radicals. The mission of the Resource is to serve the community of EPR spectroscopists with emphasis on development of advanced EPR instrumentation and new EPR methodology. [unreadable] [unreadable] OVERALL CRITIQUE: [unreadable] TECHNOLOGICAL RESEARCH AND DEVELOPMENT [unreadable] The personnel and environment for the device-driven technology research and development are outstanding. The project on resonator development was rated outstanding and deemed to be the strongest of all the core research projects. This work is innovative, of moderate risk, but with potentially high reward. The development of the uniform field (UF) resonator is exactly the type of research at which the Center excels, and was viewed with very high enthusiasm. The UF resonator represents an important advance that could have a major impact on the field. A number of special purpose resonators will be constructed for specific projects in which improved performance could have a major impact on the quality of the measurements. These resonators incorporate many innovative ideas and promise to be of considerable practical benefit. Excellent progress has been made on the development of bimodal resonators. Overall, there is an excellent balance between development of innovative new technologies, which could have considerable impact on the long-term development of the field, and construction of resonators needed for specific projects. [unreadable] [unreadable] The project on W-band engineering enhancements raised a number of concerns. The panel considered that there is an outstanding case for addition of a W-band spectrometer to the instrumentation at the Center. However, they had major concerns about the proposal to enhance the instrument. Most of these enhancements were conceived to be implementations of prior art. At the reverse site visit, the PI proposed a major change in strategy for enhancement of this instrument, which will now be based on frequency translation from Q-band (rather than X-band as described in the proposal). Insufficient details were provided to assess the feasibility of achieving adequate W-band microwave power. A major deficiency in the proposal is its failure to build on relevant prior art. Alternative solutions to many of the technical and engineering problems are available and have already been implemented in other W-band spectrometer designs. Introduction of TLSS detection at W-band would represent a major innovation; apart from this, however, the overall level of innovation in this core project is modest. There were concerns about whether the Bruker magnet is the best choice. [unreadable] [unreadable] Digital receivers could well be the way of the future. The development of broadband TLSS receivers is therefore of outstanding importance and promises to have very high impact in the field. In contrast, the benefits of broadband data streaming are unclear. This part of this core project is lacking in innovation and its potential impact on the field is questionable. [unreadable] [unreadable] The four methodology development driven core research projects were reviewed with mixed enthusiasm and it was considered that two of them lacked the level of technological development and innovation required for core research. [unreadable] [unreadable] The development of high frequency parallel mode EPR is important and could greatly facilitate analysis of paramagnetic metalloproteins with integer spin. The approach is straightforward and the resonator will be constructed using established technology. Overall, this core project was deemed to be significant and feasible but technological innovation is modest. [unreadable] [unreadable] The project on site-directed spin labeling has two components, development of saturation recovery methodology and development of spin labeling methods for W-band. The proposed research is a sound and logical extension of current spin labeling technology. However, novel technology drivers are missing. Although this is an important technique and meritorious science, the overall level of methodology development is insufficient for a core project. This core project would be better regarded as collaborative research and was therefore not assigned a score. [unreadable] [unreadable] The science behind the project on development of X-band/Q-band spin trapping methodology is excellent. There is considerable innovation in spin trap development. The synergy between spin trap design and the move to high frequency EPR, together with the implementation of higher volume resonators, will have a major and broad impact on the methodology of spin trapping. The research was poorly documented in the proposal, but more detailed information was presented at the reverse site visit which significantly increased the enthusiasm for this project. [unreadable] [unreadable] The acquisition of rapid stopped flow kinetic EPR capabilities is a highly desirable goal and a number of applications can be envisaged that would benefit from this technology. A weakness is that there appears to be no compelling application driving the technology development and the extent of technological innovation is low. The goals will be achieved largely by combining existing technologies, coupled with design of an improved resonator. The level of technological development is insufficient for a core project and it would be better regarded as collaborative research. This core was not scored. [unreadable] [unreadable] COLLABORATIVE RESEARCH [unreadable] While much of the science described in the six collaborative research projects is meritorious, only two were reviewed with high enthusiasm as drivers of core TR&D. [unreadable] [unreadable] Site-directed spin labeling of NADPH-cytochrome P450 reductase. The goals of this collaboration are to define the structure of an N-terminal domain and to probe movements between domains in the presence and absence of ligand. This was deemed to be a good application that benefits from the instrumentation of the Center and the new technologies being developed. [unreadable] [unreadable] Formation and characterization of cholesterol-glycosphingolipid raft-domains in unsaturated phosphatidylcholine membranes. Insufficient detail was provided to evaluate how this project drives or benefits from the core technologies. [unreadable] [unreadable] Low frequency L-band EPR of copper (II) in prion protein and in prion octapeptide repeats. This project was reviewed with only moderate enthusiasm since its principal impact on the core technology seems limited to driving construction of a large volume L-band resonator. [unreadable] [unreadable] Spin trapping studies of isoniazid-derived free radicals. This project seems to utilize rather than push the development of new core technologies. [unreadable] [unreadable] Characterization of the iron-sulfur centers of the bi-directional hydrogenase Cpl from Clostridium pasteurianum. This is a new collaboration introduced in the revised proposal and was viewed with high enthusiasm. [unreadable] [unreadable] The solution structure of visual arrestin. This project was reviewed with high enthusiasm as an excellent example of the use of spin label EPR that takes full advantage of the multiple quantum capabilities of the Center. [unreadable] [unreadable] SERVICE [unreadable] The previous review panel pointed out deficiencies in the presentation of the service component that were only partly addressed in the revised proposal or the reverse site visit. Service activities should be a strong component of the operations of the Center and the reviewers were concerned by the relative paucity of information and documentation about the nature and extent of services performed. They consider, as did the previous review group, that the service activities need to be strengthened to make the new technologies developed at the Center more widely available. [unreadable] [unreadable] TRAINING [unreadable] In general, the training program is excellent. The Center offers two-week training programs for young investigators and excels in training at the graduate student and postdoctoral level. [unreadable] [unreadable] DISSEMINATION [unreadable] Dissemination of technology and expertise to the community is excellent overall. The publication record is outstanding and the staff of the Center are frequently invited to speak at meetings. Problems with the web site persist. [unreadable] [unreadable] TECHNOLOGICAL RESEARCH AND DEVELOPMENT [unreadable] Priority Score: 130 [unreadable] [unreadable] D.1.1 Resonator Development: [unreadable] Description: This core project focuses on state-of-the-art EPR microwave resonator development, with an emphasis on loop gap resonators, partitioned bimodal resonators and uniform field (UF) resonators. Also emphasized in this core is the development of resonators for specific applications and collaborations, including Q-band loop gap resonators (LGR) for multiquantum experiments on aqueous samples, L-Band LGR's for low concentration frozen copper samples, large sample access TE011 Q-band cavity, parallel resonators for Q and W-bands, X-band LGR's for small aqueous samples in the context of spin-trapping, and W-band resonators for aqueous samples in the context of site-directed spin labeling (SDSL). Design and development is greatly facilitated by computer simulations of microwave field distributions in prospective designs using advanced HFSS simulation software. At the reverse site visit, the PI presented a new design for a frequency-tunable TE102-geometry cavity directly arising from simulations using this software. [unreadable] [unreadable] Significance: The development of novel EPR resonance structures and their implementation in meaningful experiments have been core strengths of the National Biomedical EPR Research Resource Center since its inception. No other commercial or academic program can compare to the scope of research activity dedicated to developing and improving continuous wave (CW) EPR resonators. The project described in the proposal is an attractive blend of state-of-the-art development of new resonators and the adaptation of existing resonator designs driven by needs encountered in the experimental collaborations. Both of these aspects were very well-received by the review panel at the previous site visit presentation, but only the former was well-described in the written proposal. Now, both the pure R&D and the experimental adaptations of resonators are detailed in the application, and improvements to both have been introduced, relative to the previous submission. This illustrates that the Center is focused, not only on the theory and design of new resonant structures, but also on their implementation in collaborative research. [unreadable] These new resonators described in this core are intended to carry out specialized experiments for which existing resonators are deficient in sensitivity and/or H1 homogeneity. Gains in sensitivity of even factors of two or three will make crucial differences between success and failure in the research. Success in these designs will prove to be highly significant in the execution of EPR experiments, in general, and for the proposed collaborations, in specific. [unreadable] [unreadable] Approach: This core project has three major parts. The first is the production of resonators designed to meet the needs of specific collaborations. There are six special purpose resonators that will be constructed to support specific, well-defined needs of particular collaborations. The resonators cover a wide range of frequencies from a few gigahertz to 95 GHz and address the unique challenges and limitations at each target frequency. In particular, this will be the first application of the immense expertise of the Center to the challenges encountered at 95 GHz. It is proposed to develop a resonator for aqueous samples in the context of site-directed mutagenesis. Specific problems with current resonators from Bruker BioSpin are identified and plans to circumvent them are proposed. This should have a major impact on the EPR of site-directed mutants, since a practical resonator for aqueous samples would allow the use of analytical purification methods to prepare the minute amounts required at 95GHz from small volumes of poorly-expressing cultures. However, no discussion is presented regarding earlier extensive results in this area resulting from several laboratories worldwide. Moreover, no mention is made of the semi-commercial 95 GHz resonator from the Ukraine that is presently in use in at least 1/3 of the labs in the world working at 95 GHz. The Ukrainian design avoids some of the problems identified with the Bruker resonator, and the fact that it is ignored, raises some concern that the Center will be duplicating and not building on current art in this particular subsection of the core. This general comment also applies to the parallel mode resonators at Q and W band, where significant progress has been made in other laboratories which has not been thoroughly considered in this proposal. [unreadable] The four other collaboration-driven resonator design projects constitute an outstanding use of the Center's expertise and resources. This section of the core project translates the achievements in resonator technology from previous projects into practical devices for specialized but important niche applications at the Center and at other labs. This is an outstanding and appropriate application of Center resources. [unreadable] The second part of this core project is the development of the uniform-field microwave cavity modes that were discovered recently by the Center. This is a new mode that offers a very uniform field over the entire length of a sample and has the promise of delivering much greater sensitivity, and in pulsed EPR applications, much greater microwave field homogeneity than any existing design. Pulsed EPR has been plagued because the excitation conditions vary across any finite-sized EPR sample, greatly reducing sensitivity because of non-optimal excitation conditions and complicating analysis, since each part of the sample experiences a different set of excitation conditions. One of the keys to the great successes of modern NMR has been their uniform fields achieved by the use of saddle coils and modified solenoid coils, and this resonator could deliver the same benefits to EPR. This is highly innovative work which involves the development of a detailed theoretical understanding of the uniform field mode using HFSS, followed by the translation of that knowledge into practical resonators for EPR applications. Two important applications are described: a high sensitivity resonator for line samples in conventional EPR tubes and a rectangular resonator for liquid, aqueous samples. Experiments run on aqueous samples in flat cells will benefit from the uniform field mode, because all points of the flat cell will be at the optimum thickness for maximum sensitivity. This degree of optimization is impossible in conventional TE102 cavities. [unreadable] The UF resonator projects will benefit from the immense theoretical and practical expertise at the Center and are an outstanding focus of the Center's resources. Significant progress in this design has been achieved in the past year in the form of the generalized Uniform Field resonator. This new UF resonator eliminates the dielectric end pieces and results in higher Q values, tunability, and the elimination of background signals from dielectrics. In addition, a prototype UF resonator has been constructed and the field homogeneity has been mapped experimentally. As predicted from the HFSS calculations, the field homogeneity is significantly improved over a much greater length in comparison to standard commercially available resonators. [unreadable] The third portion of this core project involves the development of bimodal loop-gap and cavity resonators. This is an innovative design, incorporating basic principles developed in this and other labs. These principles involve the use of highly balanced bridge designs incorporating two resonant devices to achieve a broadband balance. These earlier efforts showed some success in reducing source noise and ringing after pulsed excitation, but they were largely impractical because of limitations in source power, the lack of low-noise amplifiers and the difficulty in balancing the bridge. This revised application shows an awareness of the potential problems that will be faced and presents several ingenious ideas for conquering the problems of resonator tuning. Achieving a high balance over a broad bandwidth using two or more adjustments will be challenging, but the Center is probably the only place with the technical resources to develop a systematic method for tuning such a bimodal resonator. Such a resonator will have a great impact on the measurement of relaxation times in solutions and will provide a major performance boost for the site-directed spin labeling applications involving pulsed ELDOR or T1 measurements. This portion of the core is greatly improved over the original submission. It is an outstanding focus for the Center's efforts and promises major advances in resonator technology for field pulsed EPR. [unreadable] [unreadable] Innovation: The overall level of innovation in this core is extremely high. As stated in the significance section, this lab's expertise in resonator design and development is among the world's best. [unreadable] [unreadable] Investigator: Dr. Hyde is a world leader in instrumentation research and development in EPR spectroscopy. He has over 40 years experience in the field and has numerous technical achievements to his credit, many of which were achieved through work supported by the National Biomedical EPR Research Resource Center. No one is better qualified to lead this research effort. [unreadable] Dr. Froncisz will also be responsible for this core project. He has a proven record of resonator and instrumentation development. The contributions he has made through his developments of loop gap resonators have greatly benefited the EPR community. There is every reason to expect that these acheivements will continue with the proposed partitioned bimodal loop gaps and the uniform field resonators. [unreadable] Dr. Richard Mett is an Assistant Professor at the Milwaukee School of Engineering, who holds three degrees and nine patents in the area of electrical engineering. Mr. James Anderson is a well-establshed EPR senior technician with extensive experience in EPR instrumentation. This team is well-qualified to execute the work described in this core project. [unreadable] Each collaboration-driven project, has associated with it, scientists who are experts in a particular application. This is an outstanding example of core technology being driven by collaborations. The involvement of these collaborators will contribute to the synergy between development and application. [unreadable] [unreadable] Environment: The facilities available at the Biophysics Research Institute are outstanding. Those relevant for this project include 20,000 square feet of space, six EPR labs, an engineering complex, a microwave lab, and a machine shop. [unreadable] [unreadable] Overall Evaluation: This core project is an outstanding and innovative use of the Center's expertise. It will result in significant advances in EPR technology and generated significant enthusiasm. [unreadable] [unreadable] D.1.2 W-Band Enhancement [unreadable] Priority Score: 220 [unreadable] [unreadable] Description: The work is aimed at obtaining a commercial, turn-key W-band EPR instrument from Bruker, then enhancing its performance, ease of use, and capabilities. The enhancements entail: a) introducing TLSS detection and multiquantum, ELDOR and SR spectroscopies at W-band, via frequency-translation from an existing Q-band bridge; b) associated introduction of an improved AFC system that will compensate for field/time dependent phase drift; c) improvement of SNR, which is "compromised" in the Bruker system, through the introduction of a commercial W-band LNA; d) redesign of the resonator (Sec. D.1.1.1.) and modulation coil assembly to facilitate ease of use especially in liquids experiments. [unreadable] [unreadable] Significance: Over the past decade, there has been a surge of interest in EPR spectroscopy at W-band and higher frequency ranges. Applications of high-frequency EPR encompass a broad spectrum of biological systems; e.g., endogeneous and probe free-radicals and metal centers. The proposed technological developments are very likely to deliver the sought after enhancements in performance, ease of use, and capabilities of the Bruker W-band EPR instrument. The significance of the work will be primarily felt by: a) several high-frequency EPR spectroscopists in the USA (and more in Europe) that have acquired this instrument; b) users of these instruments at the Center. [unreadable] [unreadable] Approach: While the specific ideas to be pursued and the approaches used in pursuing them are sensible, a crucial aspect of the approach is the decision to feature the Bruker system as the starting point for these studies. The question had been asked previously: In light of the well-argued need for improvement, why feature this instrument? The PI gives a clear response to this question in the Introduction to the revised proposal: "We considered no other instrument." Had this customary analysis of available options been done, the PI would realize that other systems have been implemented, with some commercial availability, that directly address the design elements that are in need of improvement in the Bruker instrument (e.g., ease of sample access, resonator rotation, frequency stability), and that also feature frequency-translation. Included in the rationale of this choice is the issue of price, but without a comparison to any alternatives, this issue, like issues of commercial availability, ease of operation and sensitivity, are moot. Thus, the case for justifying this crucial decision has not been made by the PI. The lack of attention to prior art is most troubling in the discussion of instrument enhancement. The resonator described in D.1.1.1., is essentially the same as that which Lebedev made some 20 years ago; its most recent description is in ref. 57. Notably, this reference describes an elegant coupling scheme that provides for quick sample change, rotation of cavity orientation, and variability in coupling. The introduction of these features is a goal of the proposed research. Accordingly, the lack of an appropriate discussion of this prior art is a shortcoming of the proposal. The choice of magnet systems also appears to suffer from a similar lack of planning. For metallo-protein EPR, a sweep coil range of 700 G is unlikely to be generally satisfactory; present experience suggests that sweep of the main coil in the proposed system will lead to an unacceptable consumption of liquid helium. Arguably better options are commercially available. The PI needs to consider other options and provide a better justification of the choice made. Finally, the panel found the idea of upconversion of a Q-band to W-band by means of untested mixers, the specifications of which were not provided; to be a risky strategy that again needs to be carefully considered against proven alternatives. Overall, the lack of consideration of these issues, that are based on an apparent under-appreciation of the abundance of practical experience with high-frequency EPR that has been gained in other laboratories, does not give a sense that the program has been carefully thought out for its greatest possible scientific, technological, or service impact. [unreadable] [unreadable] Innovation: Certain aspects, in particular, the implementation of TLSS detection and mutliquantum spectroscopy are highly innovative and scientifically very attractive. Overall, however, the innovation is modest. Many of the problems considered here have already been addressed in other W-band instrumentation in place elsewhere. There is surprisingly little consideration given to this prior art in the proposal. [unreadable] [unreadable] Investigator: Dr. Hyde is the world leader in research on the development of EPR instrumentation, with over 40 years of experience that has led to over 300 publications and numerous major awards. Dr. Hyde has assembled a team of four engineers, two electronics technicians, and one machinist at the Center. Past performance indicates that this team has all of the expertise required to complete the proposed work. [unreadable] [unreadable] Environment: The strongest aspect of this core is the established, outstanding technical capabilities of the Center. [unreadable] [unreadable] Overall Evaluation: The proposed work will be conducted with exquisite skill. The inherent logic of establishing a W-band instrument for community use at the Milwaukee center, is an additional strength of this proposal. The weaknesses detailed above, however, limit enthusiasm for the proposed work. [unreadable] [unreadable] D.1.3 Broadband Digital Detection, Signal Analysis and Archiving in EPR Spectroscopy [unreadable] Priority Score: 160 [unreadable] [unreadable] Description: This section of the core research program will strive to fully characterize the digital detection methodologies of Time-locked sub-sampling (TLSS) and Time-locked over sampling TLOS for EPR applications. The PI and his research staff have incorporated these signal digitization schemes into their recently completed multiquantum Q-band spectrometer and shown that the method, which is used commercially by General Electric in their magnetic resonance imaging equipment, can be successfully used to obtain the EPR spectrum of a concentrated spin-label sample. The sampling methods used allow simultaneous detection of absorption and dispersion signals using single mixer detection. When magnetic field modulation is used to "package" the signals, one can simultaneously obtain first- through nth derivative spectra (fourth derivative spectra of a spin-label sample were demonstrated in the initial publication of this technique by the Hyde group). The PI argues that these detection methods, TLSS and TLOS, yield EPR data in their "purest" form and should constitute the method of choice for data acquisition and analysis in EPR. Experiments proposed in this section of the research plan will be aimed at characterizing performance for the purpose of microwave bridge/receiver control, exploring schemes for data storage, applying digital sampling methods for evaluating spectrometer noise performance, characterizing different paths for data processing and finally, comparing overall performance of the digital detection methods to standard schemes. [unreadable] [unreadable] Significance: These sampling schemes allow the data to be digitized directly off of the microwave mixer. The opportunity to apply different data processing and filtering schemes to EPR data after collection is an important advantage, as is the ability to select spectral displays for various modulation harmonics. Equally important, may be the use of these digital detection strategies in instrument control and in the detection of MQ-EPR. This latter capability will provide a much-needed alternative to magnetic field modulation at W-band. [unreadable] [unreadable] Approach: The characterization of TLSS and TLOS detection schemes for EPR, MQ-EPR and ELDOR experiments will be carried out on the MQ Q-band instrument mentioned above. Initial studies will explore characteristics of the intermediate-frequency modulation used, so that receiver noise can be optimized. A digital AFC system, based on detection of AFC modulation components using TLSS, will be implemented as a first project to apply these methods for microwave bridge optimization. This undertaking may be essential, as the heterodyning scheme will be very sensitive to bridge balance and drift, as compared to the conventional homodyne method. For a very similar reason, automatic control of the cavity matching (iris) adjustment may be undertaken if bridge drift is a problem. This is the logical place to start these experiments because bridge stability will be key to using the TLSS and TLOS methods on dilute samples. [unreadable] [unreadable] In addition to exploring the above issues of spectrometer control, data analysis and storage schemes will also be explored. The reviewers disagree with the supposition that the raw data for EPR spectra, including all the modulation sidebands, will, in the future, be streamed to disk for storage. Most EPR spectra are routine, and the derivative or integrated spectrum is all that is desired. The storage of raw data might be prudent with a few difficult experiments or those with the highest information content, but in that case, this kind of storage is not specific for EPR. Digital oscilloscopes now do this type of storage with, perhaps, not as deep a memory, but which can be added separately. But for buffering, compressing (or decimating), and storing such data on the fly, the Center should look to commercial markets for this general solution, which is also used in TOF-MS, as well as general electronic testing. [unreadable] [unreadable] Finally, a comparative study of digitally-detected EPR and ELDOR spectroscopies of nitroxides, with data collected using conventional homodyne phase sensitive detection schemes, will be carried out. There was some concern that these characterization experiments were less sophisticated than what one would expect, given the years of experience this Center has in MQ-EPR. However, the experiments described are logically planned and necessary. [unreadable] [unreadable] Innovation: Dr. Hyde and his coworkers are bringing important methodology from NMR imaging to EPR. This project is innovative, in that their work marks the first attempt at this type of digital sampling in cw-EPR and it will likely have significant impact. [unreadable] [unreadable] Investigator: Dr. Hyde is well-qualified to lead this effort. [unreadable] [unreadable] Environment: The environment at MCW is ideal for this project. [unreadable] [unreadable] Overall Evaluation: Digital detection is a welcome addition to cw-EPR spectroscopy that will ha