Arbitrary Pulse Shaping to Advance Electron Paramagnetic Resonance Tools for Biom

描述（由申请人提供）：将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，这将导致广泛的和根本性的重要进展，包括实现具有高光谱分辨率的傅立叶变换EPR和时间分辨EPR光谱。内置在脉冲EPR仪器中的动态核极化模块将利用这种新的脉冲成形能力，实现新一代的定量和时间分辨水合水扩散动力学测量，这些水合水是润滑蛋白质和膜系统的外部和内部的，具有特定位点的分辨率。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，商业光谱仪将提供该系统的核心。尽管最先进的EPR仪器提供了各种可定制的软件配置，但它们无法像核磁共振（NMR）光谱和成像（MRI）那样常规地形成单个脉冲。尽管形状脉冲已经在核磁共振中实施了25年以上，并且在所有临床MRI扫描仪中常规实施，但事实上，在GHz及更高频率的EPR仪器中，从未报道过任意脉冲整形的应用。提出的开发将利用最先进的技术，利用集成电路组件产生~10 GHz的数字波形，其幅度和相位可以指定为0.25-1 ns分辨率。该技术的预期优点是广泛而重要的，包括推进蛋白质，核酸，组装或脂质膜系统的结构，动力学和功能的研究。对膜蛋白（包括G蛋白偶联受体（GPCR））的纳米尺度距离测量的灵敏度将显著增强。gpcr配体激活后的关键构象变化可以通过改进的时间和光谱分辨率进行探测。在ms时间尺度上的构象动力学对酶的功能至关重要，可以量化和比较活性和非活性状态，同时探测构象亚态。有了这些能力，就可以通过调节蛋白质构象和水合动力学，直接探测酶的活性来有效地绘制药物效应。新仪器还将使研究与神经退行性疾病有关的蛋白质的早期聚集事件成为可能，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-突触核蛋白。一个关键的早期事件是蛋白质单体的（错误）折叠，这被认为是模板聚集。在聚集的早期阶段形成的可溶性蛋白低聚物已被发现具有关键的神经毒性作用，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，潜在的药物、抑制剂或突变对这些通常无法被现有工具检测到的关键物种的形成或消失的影响，以及它们对聚集的限速步骤的影响，都可以被探测到。这些进展解决了生物医学研究中的几个关键障碍。

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.