Development of Biomedical EPR Instrumentation

描述（由申请人提供）：已经开发了94 GHz （w波段）高微波频率的电子顺磁共振（EPR）光谱的惊人新颖仪器。本提案的广泛长期目标是建立该仪器在重要的生物医学应用中的实用性：使用含胆固醇的二myristoylphosphatidycholine （DMPC）膜在相图中研究胆固醇介导的脂质相互作用。磷脂，以及胆固醇，在一些可用的位点上使用14N或15N同位素进行自旋标记。在14n标记位点和15n标记位点之间发生的双分子碰撞率的测量提供了有关胆固醇对分子动力学影响的信息。实验方法的时间尺度为10¿s数量级，这是自旋标记的自旋晶格弛豫时间T1的典型值。在快10倍到慢10倍的范围内进行测量是可以实现的。这一范围被认为不仅具有高度的生物学相关性，而且使用其他仪器方式基本上无法进入。将使用两种互补的EPR技术：饱和恢复（SR）和脉冲电子-电子双共振（ELDOR）。这两种技术的激发都是在EPR谱的选定区域内对强微波频率进行绝热快速扫描。这是一个CHIRP扫频脉冲。这是一种高度创新的技术方法，需要使用任意波形发生器（AWG）和宽带环隙谐振器（LGR）。具体目标如下：(1)发展CHIRP激发和SR观测方法，然后利用可用的自旋标签测量相图的实验参数。(2)利用Specific Aim 1的数据设计类似的ELDOR实验，这也将在相图中应用不同的自旋标签对——一个带14N，一个带15N。测量不同深度标签之间的碰撞频率——所谓的“垂直波动”——将有助于深入了解胆固醇对膜动力学的中介作用。锥内扩散模型将使用14N/15N对进行测试，对中的每个成员在相同的深度，作为增加深度的函数，在相位图中。(3)，这是一项激烈的最先进的工程倡议：在现有仪器中直接数字检测微波载波。Aim 3的一个新方面是使用SR和ELDOR信号的幅度检测，这在EPR的背景下是新的，尤其适用于使用CHIRP脉冲。这一建议基于一个非常重要的假设，即分子结构需要分子动力学知识才能具有生物学相关性。可以预见，该方法将扩展到膜结合蛋白。

DESCRIPTION (provided by applicant): Strikingly novel instrumentation for electron paramagnetic resonance (EPR) spectroscopy at the high microwave frequency of 94 GHz (W-band) has been developed. The broad long-term objective of the present proposal is to establish usefulness of this instrument in a significant biomedical application: the study of cholesterol-mediated lipid interactions using dimyristoylphosphatidylcholine (DMPC) membranes containing cholesterol across the phase diagram. Phospholipids, as well as cholesterol, spin-labeled at a number of available sites using either 14N or 15N isotopes will be used. Measurements of bimolecular collision rates that occur between a 14N-tagged site and a 15N-tagged site provide information about the impact of cholesterol on molecular dynamics. The time scale for the experimental methods is on the order of 10 ¿s, which is a typical value for the spin-lattice relaxation time, T1, of the spin label. Measurements in the range of 10 times faster to 10 times slower are within reach. This is a range that is considered not only to be of high biological relevance but also to be essentially inaccessible using other instrumental modalities. Two complementary EPR techniques will be used: saturation recovery (SR) and pulse electron-electron double resonance (ELDOR). Excitation in both techniques will be an adiabatic rapid sweep of an intense microwave frequency across a selected region of the EPR spectrum. This is a CHIRP frequency-swept pulse. It is a highly innovative technical approach that requires the use of an arbitrary waveform generator (AWG) and a broadband loop-gap resonator (LGR). Specific aims are as follows: (1) Development of CHIRP excitation and observation methods for SR followed by measurements across the experimental parameters of the phase diagram using available spin labels. (2) Use of the data of Specific Aim 1 to design analogous ELDOR experiments, which will also be applied across the phase diagram using various spin-label pairs-one with 14N and one with 15N. Measurements of collision frequencies between labels at different depths-so-called "vertical fluctuations"-will be made to provide insight into the mediation of membrane dynamics by cholesterol. The diffusion-in-a-cone model will be tested using 14N/15N pairs, each member of the pair at the same depth, as a function of increasing depth, across the phase diagram. And (3), which is an intense state-of-the-art engineering initiative: direct digital detection of the microwave carrier in the existing instrument. A novel aspect of Aim 3 is the use of magnitude detection of SR and ELDOR signals-which is new in the context of EPR and is particularly appropriate when using CHIRP pulses. This proposal rests on the highly significant hypothesis that molecular structures require knowledge of molecular dynamics to be biologically relevant. Extension of the methodology to membrane-bound proteins is foreseen.