NMR Technologies for Integrating Structure, Function and Disease

经修订的申请简介 A0补助金于2019年5月提交，在亚历山大巴恩斯教授搬到苏黎世联邦理工学院之前， 乍得Rienstra被招募到威斯康星大学麦迪逊分校。现在Rienstra正式成为威斯康星大学麦迪逊分校的正教授， 已经谈判了在固态核磁共振（SSNMR）计划在NMRFAM（新的和/或移动的主要投资 来自Illinois），包括三个屏蔽的600 MHz磁体，一个750 MHz宽孔磁体，四个光谱仪， 几个定制设计的600-750 MHz魔角旋转（MAS）探头，并升级到900 MHz 光谱仪，这立即对2019年底DBP 6和其他 2020年初进行中的实验。 此外，国家科学基金会中型研究基础设施-2提案“网络， 高级NMR”，由UW-Madison PI（Rienstra和Henzler-Wildman）合作提交 与康州大学健康中心的杰夫·霍克和美国大学的艺术爱迪生一起。格鲁吉亚正处于与 NSF。如果获得资助，这笔赠款将在2022-23年为NMRFAM带来1.1 GHz专用SSNMR光谱仪。 这些发展促使对A1应用程序进行了几项更改， NMRFAM的SSNMR程序： (1)Rienstra现在是PI和Henzler-Wildman的共同PI。 (2)TR&D1现在包括针对微型转子包装和样品操作开发的子目标 利用最近在转子直径<1 mm的超快MAS（>100 kHz）方面的突破的工具; 扩大了TR&D 1的范围和影响。还提供了梯度下NMR的基线和基准 更明确的描述和原理验证实验已经到位。 (3)TR&D2现在解决了SSNMR数据收集中的关键瓶颈，强调： (a)参数优化和光谱仪配置的自动化; (b)600-900 MHz的新窄孔魔角旋转探头设计（适用于1.1 MHz GHz及未来更高）;以及 (c)与数据处理的实时反馈交互（TR&D3）。 (4)TR&D3现在利用NMRFAM软件产品和持续的技术开发来实现稳固的 国家NMR，包括分配，结构测定，完善和验证工具，它是更多 与提案的其余部分完全融合。 (5)DBPs 5、6和7已更改，以包括以下方面之间发展良好且有影响力的合作： Rienstra和Paul Kotzbauer（华盛顿州）联合医学，路易体和突触核病），马蒂伯克（美国。 伊利诺斯州，抗真菌药物）和詹姆斯莫里西（美国。Michigan，血液凝固）。 总的来说，现在的提案（我们认为）是以一种更符合逻辑/时间顺序的方式组织的，TR&D1 TR&D2侧重于样品，TR&D3侧重于软件和分析程序。 提案主体进一步解释了这些发展如何大大提高项目的成本效益比 并与当前的用户计划和NMRFAM的长期愿景相结合。 这些修订还解决了A0申请审查人员的首要问题，包括“狭窄”， TR&D1的范围/适度创新”;“TR&D3的弱整合”;成本效益比;初步数据;解决方案 vs. TR&D3的重点;创新;背景（解决相互竞争的想法和先例）;和特殊性 的结果。最后，我们阐明了关于膜不对称性的前提和途径 proteins.

INTRODUCTION TO REVISED APPLICATION The A0 grant was submitted in May 2019, before Prof. Alexander Barnes moved to ETH-Zurich and before Prof. Chad Rienstra was recruited to UW-Madison. Now Rienstra is officially a Full Professor at UW-Madison, after having negotiated major investments in the solid-state NMR (SSNMR) program at NMRFAM (new and/or moving from Illinois) including three shielded 600 MHz magnets, one 750 MHz wide bore magnet, four spectrometers, several custom-designed magic-angle spinning (MAS) probes at 600-750 MHz, and upgrades to the 900 MHz spectrometer, which immediately have had an impact on data collection for DBP6 in late 2019 and for other experiments in progress during early 2020. Furthermore, the National Science Foundation Mid-Scale Research Infrastructure-2 proposal on "Network for Advanced NMR", which was submitted by UW-Madison PIs (Rienstra and Henzler-Wildman) in collaboration with Jeff Hoch at UConn Health and Art Edison at U. Georgia, is in late stages of negotiation and review with NSF. If funded, this grant would bring a 1.1 GHz dedicated SSNMR spectrometer to NMRFAM in ~2022-23. These developments have motivated several changes to this A1 application which more explicitly emphasizes the SSNMR program at NMRFAM: (1) Rienstra is now contact PI and Henzler-Wildman co-PI. (2) TR&D1 now includes sub-aims targeting development of micro-rotor packing and sample manipulation tools to leverage recent breakthroughs in ultra-fast MAS (>100 kHz) at <1 mm rotor diameters; this broadens the scope and impact of TR&D 1. Baselines and benchmarks for NMR under gradients are also more clearly described and proof-of-principle experiments are in place. (3) TR&D2 now addresses critical bottlenecks in SSNMR data collection, emphasizing: (a) automation for parameter optimization and spectrometer configuration; (b) new narrow bore magic-angle spinning probe designs at 600-900 MHz (that will be applicable at 1.1 GHz and higher in the future); and (c) real-time feedback interaction with data processing (TR&D3). (4) TR&D3 now leverages NMRFAM software products and continuing technology development for solid- state NMR, including assignment, structure determination, refinement and validation tools, and it is more clearly integrated with the rest of the proposal. (5) DBPs 5, 6 and 7 have been changed to include well-developed and impactful collaborations between Rienstra and Paul Kotzbauer (Wash. U. Medicine, Lewy bodies and synucleiopathies), Marty Burke (U. Illinois, antifungal drugs), and James Morrissey (U. Michigan, blood coagulation). Overall the proposal is now organized in (we think) a more logical/chronological manner, with TR&D1 emphasizing samples, TR&D2 the spectrometer and probes, and TR&D3 the software and analysis procedures. The proposal body further explains how these developments greatly augment the cost-benefit ratio for the project and integrate with the current user program and the long-term vision of NMRFAM. The revisions also address the overarching concerns of reviewers of the A0 application including "narrow scope/modest innovation in TR&D1"; "weak integration of TR&D3"; cost-benefit ratio; preliminary data; solution vs. solids emphasis of TR&D3; innovation; context (addressing competing ideas and precedents); and specificity of outcomes. Finally, we have clarified the premise and approach with respect to asymmetry in membrane proteins.