Arbitrary Pulse Shaping to Advance Electron Paramagnetic Resonance Tools for Biom

任意脉冲整形促进 Biom 电子顺磁共振工具的发展

• 批准号: 8298123
• 负责人: Songi Han
• 金额: $ 15.51万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-15 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8298123
• 关键词: Address; Alzheimer' s Disease; Amyloid; Binding; Biochemical; Biomedical Research; Clinical; Communities; Computer software; Coupled; Custom; Detection; Development; Disease; Drug Delivery Systems; Electron Spin Resonance Spectroscopy; Electrons; Enzymes; Event; Fiber; Fourier Transform; Frequencies; G-Protein-Coupled Receptors; Heating; Hydration status; Image; Individual; Kinetics; Lead; Ligands; Magnetic Resonance Imaging; Maps; Measurement; Membrane; Membrane Lipids; Membrane Proteins; Methodology; Molecular; Molecular Conformation; Mutation; NMR Spectroscopy; Neurodegenerative Disorders; Nuclear; Nuclear Magnetic Resonance; Nucleic Acids; Parkinson Disease; Pharmaceutical Preparations; Phase; Physiologic pulse; Population; Preclinical Drug Evaluation; Protein Conformation; Proteins; Pump; Reporting; Resolution; Sampling; Shapes; Signal Transduction; Site; Solutions; Source; Specific qualifier value; Specificity; Spectrum Analysis; Staging; Structure; Surface; System; Techniques; Technology; Time; Training Programs; Ursidae Family; Water; alpha synuclein; base; design; digital; enzyme activity; flexibility; improved; inhibitor/antagonist; instrument; membrane assembly; microwave electromagnetic radiation; monomer; nanoscale; neurotoxic; new technology; next generation; protein aggregation; protein folding; protein function; protein misfolding; protein oligomer; protein structure; receptor structure function; response; structural biology; tau Proteins; tool; two-dimensional

DESCRIPTION (provided by applicant): For the first time, an arbitrary pulse shaping module will be integrated into an Electron Paramagnetic Resonance (EPR) spectrometer that will lead to wide ranging and fundamentally important advances, including the realization of Fourier Transform EPR with high spectral resolution and time-resolved EPR spectroscopy. A Dynamic Nuclear Polarization module built into the pulsed EPR instrument will capitalize on this new pulse shaping capability to enable the next generation of quantitative and time resolved measurements of diffusive dynamics of hydration water that is lubricating the exterior and interior of proteins and membrane systems, with site-specific resolution. These are all entirely new experimental capabilities. For the broadest possible dissemination of these novel technologies to the biomedical user community, a commercial spectrometer will provide the core of the system. Despite the variety of software-customizable configurations offered by state of the art EPR instruments, they offer no ability to shape individual pulses, as is done routinely in nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI). Although shaped pulses have been implemented in NMR for over 25 years and are routinely implemented in all clinical MRI scanners, the application of arbitrary pulse shaping has, in fact, never been reported before for any EPR instrument at GHz frequencies and higher. The proposed development will capitalize on a state of the art technique that employs integrated circuit components to generate digital waveforms at ~10 GHz, whose amplitude and phase can be specified with 0.25-1 ns resolution. The expected merits of this technology are broad and significant, and include advancing the study of structure, dynamics and function of proteins, nucleic acids, assemblies or lipid membrane systems. The sensitivity for nanometer scale distance measurements of membrane proteins, including G protein-coupled receptors (GPCR), will be significantly enhanced. Critical conformation changes upon ligand-activation of GPCRs can be probed with improved temporal and spectral resolution. Conformational dynamics on the ms timescale-critical for enzyme function-can be quantified and compared between the active and inactive state, while probing the conformational substates. Equipped with these capabilities, drug effects can be effectively mapped out by directly probing the enzyme activity, through the modulation of protein conformation and hydration dynamics. The new instruments will also enable the study of early aggregation events of proteins implicated in neurodegenerative diseases, e.g. tau and amyloid-b in Alzheimer's or a-synnuclein in Parkinson's disease. One critical early event is the (mis)folding of the protein monomer that is thought to template aggregation. Soluble protein oligomers formed in the early stages of aggregation has been found to bear critical neurotoxic effects, likely more than the fibrous aggregates. The new tools will be capable of characterizing the dynamic structure of these oligomers, and quantifying the kinetics of protein folding, oligomer formation and fiber maturation with site-specificity and high time resolution. Thus, the effects of potential drugs, inhibitors or mutations can be probed on the formation or disappearance of these critical species that usually escape the detection of existing tools, and their effect on the rate limiting step of aggregation. These advances address several critical barriers in biomedical research.

描述（由申请人提供）：任意脉冲整形模块将首次集成到电子顺磁共振（EPR）波谱仪中，这将带来广泛的、根本性的重要进步，包括实现具有高谱线的傅里叶变换 EPR分辨率和时间分辨 EPR 光谱。脉冲 EPR 仪器中内置的动态核极化模块将利用这种新的脉冲整形功能，实现对润滑蛋白质和膜系统外部和内部的水合水扩散动力学的下一代定量和时间分辨测量，特定于站点的分辨率。这些都是全新的实验能力。为了尽可能广泛地向生物医学用户社区传播这些新技术，商业光谱仪将提供系统的核心。尽管最先进的 EPR 仪器提供了各种软件可定制配置，但它们无法像核磁共振 (NMR) 光谱和成像 (MRI) 中常规那样形成单个脉冲。尽管整形脉冲在 NMR 中的应用已超过 25 年，并且在所有临床 MRI 扫描仪中常规应用，但事实上，之前从未报道过任意脉冲整形在任何 GHz 频率及更高频率的 EPR 仪器中的应用。拟议的开发将利用最先进的技术，该技术采用集成电路元件来生成约 10 GHz 的数字波形，其幅度和相位可以以 0.25-1 ns 的分辨率指定。这项技术的预期优点是广泛而重要的，包括推进蛋白质、核酸、组装体或脂质膜系统的结构、动力学和功能的研究。包括 G 蛋白偶联受体 (GPCR) 在内的膜蛋白纳米级距离测量的灵敏度将显着增强。可以通过改进的时间和光谱分辨率来探测 GPCR 配体激活时的关键构象变化。毫秒时间尺度上的构象动力学（对于酶功能至关重要）可以在活性和非活性状态之间进行量化和比较，同时探测构象亚状态。配备这些功能，可以通过直接探测酶活性、通过调节蛋白质构象和水合动力学来有效地绘制药物效应。新仪器还将能够研究与神经退行性疾病有关的蛋白质的早期聚集事件，例如神经退行性疾病。阿尔茨海默病中的 tau 蛋白和淀粉样蛋白 -b 或帕金森病中的 a-突触核蛋白。一个关键的早期事件是蛋白质单体的（错误）折叠，被认为是模板聚集。人们发现，在聚集早期形成的可溶性蛋白质寡聚物具有严重的神经毒性作用，可能比纤维聚集体更严重。新工具将能够表征这些低聚物的动态结构，并以位点特异性和高时间分辨率量化蛋白质折叠、低聚物形成和纤维成熟的动力学。因此，可以探究潜在药物、抑制剂或突变对这些通常逃避现有工具检测的关键物种的形成或消失的影响，以及它们对聚集限速步骤的影响。这些进步解决了生物医学研究中的几个关键障碍。