Acquisition of a 500 MHz NMR Spectrometer for Structural Analysis of Biological Macromolecules

我们建议通过收购最先进的600 MHz高分辨率多核核磁共振波谱仪，在康奈尔大学生物技术大楼建立一个生物分子核磁共振中心，专门用于结构生物学的研究和培训。康奈尔大学的跨学科生物物理学项目高度致力于大分子结构的研究。该小组在X射线结晶学、大分子结构和动力学的理论/计算研究以及蛋白质功能的生物化学和分子遗传学研究方面建立了国际公认的设施和人员。拟议的600 MHz核磁共振光谱仪将通过提供结构和动力学的溶液研究能力来补充现有资源。康奈尔大学的化学核磁共振设施中安装了一台500 MHz核磁共振光谱仪，它没有配备最新的技术进步(例如，第四射频通道、XYZ脉冲场梯度放大器和探头、10 mm三重共振探头和增强型室温垫片)。这台机器通常都是满负荷的，其庞大的用户群既有“小分子”用户，也有“大分子”用户。过去康奈尔大学蛋白质溶液核磁共振研究的进展主要是通过利用外部资源实现的，要么是在合作者的实验室里，要么是在公共设施中。在过去的十年中，三重和四重共振多维核磁共振波谱已经成为现代结构生物学的重要组成部分。为了跟上这一加速发展的研究领域的步伐，并为我们的本科生和研究生在这个增长最快的科学领域之一的职业生涯做好准备，必须将这项技术引入康奈尔大学。生物分子核磁共振中心的主任将是去年秋天加入康奈尔大学教职的琳达·尼科尔森教授。尼科尔森教授带来了将多维核磁共振应用于蛋白质动力学和结构研究的最新专业知识，她最近在美国国立卫生研究院进行的艾滋病毒-L蛋白酶的工作证明了这一点。她带来了应用于生物大分子的最新的双重、三重和四重共振技术的知识。这种专业知识在康奈尔大学得到了哈罗德·谢拉加和罗伯特·奥斯瓦尔德长期建立的项目的高度补充，这些项目广泛利用多维核磁共振技术研究蛋白质的结构。在这些研究小组中取得的进展只是日常指导设施将由一名科学家工作人员提供，他们将协助和培训用户，提供技术支持，并促进协作互动。核磁共振设施的外部审查将由第5d节所述的访问委员会提供。资源分配将由一个委员会决定，该委员会由主任、工作人员科学家、一名主要用户和一名其他用户组成。光谱仪时间的安排将由科学家工作人员负责。这个项目的费用分摊将由艺术与科学学院和分子结构计划提供资金。Nicholson、Oswald和Scheraga组成了主要用户的“临界质量”，将在拟议的生物分子核磁共振中心启动第一个结构和动力学研究。其他几名教员，包括Begley、Jelinski、Rodriguez和Feigenson，在溶液核磁共振光谱学方面拥有公认的专业知识，并将组成更多用户的强大核心。研究活动包括研究两种普遍存在的生物物理交换机制(蛋白质磷酸化和GTP结合，Nicholson和Oswald)，蛋白质折叠和蛋白质间相互作用(Scheraga)，将蛋白质结合在一起的基本作用力(观察压力对蛋白质柔韧性的影响，Ealick)，控制脂行为的基本作用力(Feigenson)，结构和动力学在酶催化机制中的作用(Begley)，基因转录调控的双物理基础(Lis和Tye)，高效生物聚合物的生物物理基础(Jelinski)，以及生物活性天然产物与DNA之间的相互作用(Rodriguez)。方法包括梯度增强多维实验，以获得用于结构确定的顺序分配、距离约束和二面角约束，以及用于蛋白质动力学的松弛参数。使用所要求的仪器，也可以研究较大和较难溶解的系统。除了科学研究，核磁共振设施还将通过正规课程和培训课程、研究生和研究生研究项目以及本科生的独立学习和实地考察提供教育机会。

We propose to establish a Biomolecular NMR Center in Cornell's Biotechnology Building through acquisition of a state-of-the-art 600 MHz high resolution multinuclear NMR spectrometer, to be dedicated for research and training in structural biology. The interdisciplinary biophysics program at Cornell is highly committed to studies of macromolecular structure. This group has established internationally recognized facilities and faculty for X-ray crystallography, theoretical/computational studies of acromolecular structure and dynamics, and biochemical and molecular genetic studies of protein function. The proposed 600 MHz NMR spectrometer will complement existing resources by providing capabilities for solution studies of structure and dynamics. A single 500 MHz NMR spectrometer which is not equipped with the latest technological advances (e.g. fourth radio frequency channel, XYZ pulsed field gradient amplifiers and probes, 10 mm triple resonance probe and enhanced room temperature shims) is housed in Cornell's Chemistry NMR Facility. This machine is routinely booked to full capacity with an extensive user base composed of both "small molecule" and "macromolecule" users. Past progress in solution NMR studies of proteins at Cornell has occurred largely through utilization of outside resources either in the laboratories of collaborators or at public facilities. Over the past decade, triple- and quadruple-resonance multidimensional NMR spectroscopy has become a critical component of modern structural biology. In order to keep pace in this accelerating field of research, and to prepare our undergraduate and graduate students for careers in one of the fastest growing areas of science, this technology must be brought to Cornell. The Director of the Biomolecular NMR Center will be Professor Linda Nicholson who joined the Cornell faculty last fall. Professor Nicholson brings up-to-date expertise in the application of multi-dimensional NMR to dynamical and stru ctural studies of proteins, as evidenced by her recent work on the HIV-l protease performed at the NIH. She brings knowledge of the latest double-, triple- and quadruple- resonance techniques for application to biological macromolecules. This expertise is highly complemented at Cornell by the long-established programs of Harold Scheraga and Robert Oswald that extensively utilize multi-dimensional NMR for structural studies of proteins. progress in these research groups has been made only Day to day direction of the facility will be provided by a staff scientists who will assist and train users, provide technical support and facilitate collaborative interactions. External review of the NMR facility will be provided by the visiting committee described in section 5d. Allocation of resources will be decided by a committee that consists of the director, the staff scientist, one major user and one other user. Scheduling of spectrometer time will be the responsibility of the staff scientist. Cost sharing for this project will be provided by funds from the College of Arts and Sciences and from the Program in Molecular Structure. Nicholson, Oswald and Scheraga comprise the "critical mass" of major users that will launch the first structural and dynamics studies in the proposed Biomolecular NMR Center. Several additional faculty members, including Begley, Jelinski, Rodriguez, and Feigenson, have a demonstrated expertise in solution NMR spectroscopy, and will comprise a strong core of additional users. Research activities encompass investigation of two ubiquitous biophysical switching mechanisms (protein phosphorylation and GTP binding, Nicholson and Oswald), protein folding and protein- protein interactions (Scheraga), the basic forces that hold proteins together (observing the effects of pressure on protein flexibility, Ealick), basic forces governing lipid behavior (Feigenson), the role of structure and dynamics in mechanisms of enzyme catalysis (Begley), and the bi ophysical basis of regulation of gene transcription (Lis and Tye), the biophysical basis of high-performance biopolymers (Jelinski), and interactions between bioactive natural products and DNA (Rodriguez). Methodologies include gradient- enhanced multidimensional experiments to obtain sequential assignments, distance restraints and dihedral angle restraints for structure determination and relaxation parameters for protein dynamics. Studies of larger and less soluble systems will also be possible with the requested instrumentation. In addition to scientific research, the NMR facility will provided educational opportunities through formal courses and training sessions, through graduate and post-graduate research projects and through independent studies and field trips for undergraduate students.