This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Pulsed double electron-electron resonance (PELDOR or DEER) is an established technique for the determination of distances and their distributions in low-temperature solids. The technique is based on recording the amplitude of primary or refocused echo as a function of the position of an additional microwave pulse applied at a different frequency. The technique is useful for sufficiently broad ESR spectra as the pulses at the two frequencies should have small or no overlap. Typically, it is realized with two independent pulse sources and bimodal resonators or else with a single source and low-Q dielectric or loop-gap resonator. The technique is useful when spin concentrations are not small. In some cases of spin-labeled membrane proteins reconstituted in liposomes with large excess water there are increased local spin concentrations in the lipid phase leading to a loss of the signal by the mechanism of instantaneous diffusion. Also, there are cases such as spin clusters, e.g. protein oligomers, when the use of strong pulses leads to a loss of the signal. DEER based on weak pulses is less sensitive to instantaneous diffusion and can compete favorably with DQC in such cases. In order to accommodate a wider range of systems, we set up DEER at Ku-band (17.4 GHz), by adding a second pulse channel to the existing 9/17GHz FT-ESR spectrometer currently used for DQC work. The use of a higher frequency than standard 9 GHz leads to a higher sensitivity for typical sample volumes. The pumping pulse at the second frequency is injected via a directional coupler into the path to the dielectric resonator, which is tuned for DQC and therefore has the sufficiently low Q needed to accommodate pulses with a frequency difference of 65-70 MHz. Switching back and forth between DEER and DQC can be accomplished in a minute. We have successfully applied Ku-band DEER to the study of the small heat-shock protein Hsp16.5, voltage-dependent K+ channel KvAP, and unfolded Cytochrome-c.
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