Arbitrary Pulse Shaping to Advance Electron Paramagnetic Resonance Tools for Biom

描述(申请人提供)：首次将任意脉冲整形模块集成到电子顺磁共振(EPR)光谱仪中，这将带来广泛和根本的重要进展，包括实现高光谱分辨率的傅里叶变换EPR和时间分辨EPR光谱学。脉冲EPR仪器内置的动态核极化模块将利用这一新的脉冲整形能力，以特定位置的分辨率对润滑蛋白质和膜系统外部和内部的水化水的扩散动力学进行下一代定量和时间分辨测量。这些都是全新的实验能力。为了将这些新技术尽可能广泛地传播到生物医学用户社区，商业光谱仪将提供该系统的核心。尽管最先进的EPR仪器提供了各种软件可定制的配置，但它们不能像在核磁共振(NMR)波谱和成像(MRI)中常规的那样提供对单个脉冲进行整形的能力。尽管整形脉冲已经在核磁共振中应用了超过25年，并在所有临床MRI扫描仪中常规应用，但事实上，在GHz频率和更高频率的EPR仪器中，任意脉冲整形的应用之前从未被报道过。这项拟议的开发将利用最先进的技术，使用集成电路元件生成~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显著的，包括促进蛋白质、核酸、组件或类脂膜系统的结构、动力学和功能的研究。包括G蛋白偶联受体在内的膜蛋白纳米距离测量的灵敏度将显著提高。通过改进的时间和光谱分辨率，可以探测GPCRs配体激活时的关键构象变化。在毫秒时间尺度上的构象动力学-对酶功能至关重要-可以被量化，并在探测构象亚态的同时，在活跃状态和非活跃状态之间进行比较。有了这些能力，通过直接探测酶的活性，通过调节蛋白质构象和水化动力学，可以有效地描绘药物的效果。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，例如阿尔茨海默病中的tau和淀粉样蛋白b或帕金森病中的a-突触核素。一个关键的早期事件是被认为是模板聚集的蛋白质单体的(错误)折叠。在凝聚的早期阶段形成的可溶性蛋白低聚物被发现承担着关键的神经毒性作用，可能比纤维聚集体更强。这些新工具将能够表征这些低聚物的动态结构，并以特定的位置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，可以探讨潜在药物、抑制剂或突变对这些通常逃脱现有工具检测的关键物种的形成或消失的影响，以及它们对聚集的速率限制步骤的影响。这些进展解决了生物医学研究中的几个关键障碍。 与公共卫生相关(由申请人提供)：将开发一套新的仪器功能，大大提高目前基于电子顺磁共振光谱学的主要结构生物学工具的灵敏度、选择性和时间分辨率，并解决生物医学研究进展的几个关键障碍。这将大大推进对膜蛋白结构的研究，这些结构代表了当前大多数药物靶标，促进了激活或停用重要酶的药物筛选，或有助于揭示与神经退行性疾病有关的蛋白质聚集机制对疾病的影响。

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. PUBLIC HEALTH RELEVANCE (provided by applicant): A set of new instrumental capabilities will be developed that will significantly advance the sensitivity, selectivity and time resolution of currently prominent structural biology tools based on electron paramagnetic resonance spectroscopy, and address several critical barriers to progress in biomedical research This will significantly advance the study of membrane protein structures that present the majority of current drug targets, facilitate drug screening for activating or deactivating important enzymes or help unravel the diseases effects of protein aggregation mechanisms implicated in neurodegenerative diseases.