Development of Biomedical EPR Instrumentation

描述（由申请人提供）：已开发出在94 GHz（W波段）的高微波频率下用于电子顺磁共振（EPR）光谱的引人注目的新型仪器。本提案的广泛的长期目标是建立这种仪器在重要的生物医学应用中的有用性：使用含有胆固醇的二肉豆蔻酰磷脂酰胆碱（DMPC）膜在相图中研究胆固醇介导的脂质相互作用。将使用使用14 N或15 N同位素在许多可用位点进行自旋标记的磷脂以及胆固醇。测量14 N标记位点和15 N标记位点之间发生的双分子碰撞率可以提供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是10 μ s，这是自旋标记的自旋-晶格弛豫时间T1的典型值。在10倍快到10倍慢的范围内进行测量是可以实现的。这是一个范围，被认为不仅是高度的生物相关性，而且基本上是无法使用其他仪器的方式。两种互补的EPR技术将被使用：饱和恢复（SR）和脉冲电子-电子双共振（ELDOR）。在这两种技术中，激励都是在EPR谱的选定区域内对强微波频率进行绝热快速扫描。这是一个线性调频脉冲扫频脉冲。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）发展了同步辐射的CHIRP激发和观测方法，然后利用现有的自旋标记测量了相图的实验参数。(2)使用特定目标1的数据来设计类似的ELDOR实验，该实验也将应用于使用各种自旋标记对的相图-一个具有14 N，一个具有15 N。在不同深度的标签之间的碰撞频率的测量-所谓的“垂直波动”-将提供深入了解膜动力学的胆固醇调解。将使用14 N/15 N对来测试锥内扩散模型，在相图中，该对的每个成员处于相同的深度，作为深度增加的函数。以及（3），这是一个强烈的国家最先进的工程倡议：直接数字检测微波载波在现有的仪器。Aim 3的一个新方面是使用SR和ELDOR信号的幅度检测，这在EPR的上下文中是新的，并且在使用CHIRP脉冲时特别合适。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与生物学相关。扩展的方法，膜结合蛋白质的预见。

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.