ADDING KU-BAND DEER CAPABILITY TO THE DQC SPECTROMETER

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 脉冲双电子-电子共振(PELDOR或DEER)是一种确定低温固体中距离及其分布的成熟技术。该技术基于记录作为以不同频率施加的附加微波脉冲的位置的函数的初级或重新聚焦的回波的幅度。这项技术对于足够宽的ESR谱是有用的，因为这两个频率的脉冲应该有很小的重叠或没有重叠。通常，它是用两个独立的脉冲源和双模谐振器实现的，或者用一个源和低Q介质或环隙谐振器实现的。当自旋浓度不小时，这项技术很有用。在某些情况下，在含有大量过量水的脂质体中重组的自旋标记膜蛋白，在脂相中局部自旋浓度增加，导致信号通过瞬时扩散机制丢失。此外，当使用强脉冲导致信号损失时，也存在诸如自旋团簇的情况，例如蛋白质低聚物。基于弱脉冲的鹿对瞬时扩散不那么敏感，在这种情况下可以与DQC竞争。为了适应更广泛的系统，我们在Ku波段(17.4 GHz)设置了Deer，在目前用于DQC工作的现有9/17 GHz FT-ESR光谱仪上增加了第二个脉冲通道。使用比标准9 GHz更高的频率会导致对典型样本量的灵敏度更高。第二频率的泵浦脉冲通过定向耦合器注入到介质谐振器的路径中，介质谐振器针对DQC进行调谐，因此具有足够低的Q来容纳具有65-70 MHz频差的脉冲。在鹿和DQC之间来回切换可以立即完成。 我们已经成功地将Ku-band鹿应用于小分子热休克蛋白Hsp16.5、电压依赖性K通道KvAP和未折叠细胞色素-c的研究。

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.