This application is for an upgrade to Q-band for our current Bruker X-band E580 pulse spectrometer capable of running DEER (double electron-electron resonance), DQC (double quantum coherence), and ENDOR (electron nuclear double resonance) experiments at cryogenic temperatures. The primary use of both the current and upgraded instrument will be to quantitate distance measurements between paramagnetic probes on or within biomedically relevant proteins. The major advantages of upgrading to Q-band (35 GHz) DEER from X-band (9 GHz) DEER are a >10-fold increase in sensitivity and overall higher quality distance data. The improvement in resolution, accuracy, identification, signal intensity and the collection of longer distances will be of considerable benefit to an array of biological projects. Additional advantages of pulsed Q- band over X-band include smaller sample volumes, lower overall sample concentration requirements, and significantly decreased data collection times. Pulsed Q-band ENDOR also provides technological and fundamental benefits over X-band ENDOR. The Q-band resonator allows for significantly higher radio frequency (RF) fields for a given supplied power, which is often the limiting factor in ENDOR sensitivity, and the microwave field is increased at Q-band;these allow for the study of fast-relaxing systems that are not amenable to pulsed X-band or continuous-wave ENDOR. In addition, 14N and 1H ENDOR resonances typically overlap at X-band, yet are readily assigned at Q-band. Each one of these is a remarkable advantage to biological research projects and coupled with the considerable improvement in data accuracy will have a substantial impact on current and future structural biology studies of soluble and membrane proteins and protein complexes. Frequencies higher than Q-band provide little additional benefit to the proposed experiments, thus this upgrade request is for the optimum instrument for long-term pulse EPR spectroscopy use. This type of state-of-the-art Q-band pulsed EPR instrumentation is lacking not only at the Medical College of Wisconsin but in entire the state of Wisconsin. Five major users are identified who will measure long-range distances within proteins and protein complexes. In addition, numerous minor users are identified, each with a need for advanced pulse instrumentation not available in the region. Specifically, the research benefiting from the requested upgrade will contribute to a better understanding of the physiology of disease processes such as epilepsy, arrhythmia, neurological disorders, and cancer, and to the development of novel antibiotics and cancer therapeutic agents. Additional opportunities are expected to be uncovered once the success of the initially proposed projects is evident, opening up further avenues of interdisciplinary science. This upgraded instrumentation capability will immediately and significantly advance the productivity of the projects outlined in this proposal as well as enhance the research environment for the surrounding community.
|Zhuo, Ya; Vishnivetskiy, Sergey A; Zhan, Xuanzhi et al. (2014) Identification of receptor binding-induced conformational changes in non-visual arrestins. J Biol Chem 289:20991-1002|