DEVELOPMENT OF BIOMEDICAL ESR INSTRUMENTATION

生物医学ESR仪器的发展

• 批准号: 6605819
• 负责人: JAMES S HYDE
• 金额: $ 32.24万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6605819
• 关键词: biomedical equipment development; computer program /software; computer system design /evaluation; copper; electron spin resonance spectroscopy; metal complex; metalloproteins; method development; oxygen transport

This competitive renewal proposal is to develop pulse saturation-recovery (SR) electron paramagnetic resonance (EPR) for application to spin labels in a context of site directed spin labeling (SDSL). The applicant's laboratory has recently made a breakthrough in SR detection based on a novel digital sub-sampling strategy, which has been termed time locked sub-sampling, and a new pulse spectrometer is under construction. Thorough engineering evaluation and design optimization including signal processing software are proposed for the next funding period. Feasibility for a new X-band bimodal loop gap sample resonator has also been demonstrated and further innovation is proposed. Engineering-based development follows naturally from the work of the previous funding period. SR can measure the rate of bimolecular collisions of spin labels with oxygen, and is a primary methodology for studying oxygen transport. Spin label oximetry is an important tool in identification of structural motifs such as alpha-helices or beta-sheets in proteins through SDSL methods. Three model systems are used here in each of three application-based specific aims. The model systems, in order of increasing biological relevance, are maleimide spin label in super-cooled sec-butylbenzene, hemoglobin labeled at beta93 in glycerol-water mixtures, and the ferric enterobactin receptor, Fep-A. The SR method is validated and compared with more conventional methodology. A novel SR method to separate overlapped spectra, termed discrimination by oxygen transport, is used to characterize multiple motional components of FepA. SR apparatus acquires intense free induction decay (FID) signals that are normally suppressed. Instead, this investigator proposes to use FID signals to measure site-to-site distances in double-labeled FepA. Frequency swept pulse electron-electron double resonance is introduced to characterize slow restricted angle motions in Fep-A in a situation where overall motion of the protein is negligible. The detection method, the bimodal resonator, use of FID signals for distance determination, use of SR to separate overlapped spectra, and measurement for the first time of restricted-angle motions are thought to be highly innovative. SDSL is a new and rapidly expanding methodology in structural biology. Addition of saturation recovery to SDSL methodology will, we hypothesize, revolutionize the field. The proposal lies at the foundation of modern biomedical research: development of tools that enhance one's ability to determine protein structure, dynamics and function. The investigator asserts that only on such a foundation can a rational approach to improved human health be based.

该竞争性更新建议是发展脉搏饱和恢复 （SR）电子顺磁共振（EPR）用于旋转 在站点定向自旋标签（SDSL）的背景下标记。申请人的 实验室最近取得了基于SR检测的突破 在一种新颖的数字子采样策略中，该策略被称为时间锁定 子采样和新的脉冲光谱仪正在构造中。彻底 工程评估和设计优化，包括信号处理 在下一个资金期间提出了软件。新X波段的可行性 双峰环间隙样品谐振器也已被证明，并进一步 提出了创新。基于工程的发展自然而然 上一个资金期的工作。 SR可以测量双分子的速率 自旋标签与氧的碰撞，是用于的主要方法论 研究氧运输。自旋标签的血氧仪是重要的工具 识别结构图案，例如α-螺旋或beta表 通过SDSL方法的蛋白质。这里的每个模型系统都在这里使用 三个基于申请的特定目标。模型系统按顺序 生物学相关性的提高，是超冷的MaleImide自旋标记 SEC丁基苯，在甘油 - 水混合物中标记为β93的血红蛋白，和 铁肠霉素受体FEP-A。 SR方法已验证，并且 与更常规的方法相比。一种新型的SR方法来分开 重叠的光谱（称为氧运输歧视）用于 表征FEPA的多个运动成分。 SR设备获得 通常被抑制的强烈自由感应衰减（FID）信号。 相反，该调查人员建议使用FID信号来测量站点到现场 双标签FEPA的距离。频率扫荡脉冲电子电子 引入双共振是为了表征慢速限制角度运动 在蛋白质总体运动可忽略的情况下，FEP-A。这 检测方法，双峰谐振器，使用FID信号用于距离 确定，使用SR分离重叠光谱，并测量 首次被限制的角度动作被认为是高度创新的。 SDSL是结构生物学中的一种新的且快速扩展的方法。添加 我们假设，彻底革新SDSL方法的饱和恢复 领域。该提案是现代生物医学研究的基础： 开发可以增强人们确定蛋白质结构能力的工具 动力和功能。调查人员断言，只有在这样的基础上 改善人类健康的合理方法是否可以基于。