Development of a 750 MHz NMR Spectrometer

申请70万美元的资金用于购买17.6特斯拉核磁共振磁体以及设计和建造相应的核磁共振控制台。750兆赫。我们拟建设的核磁共振控制台，将能够执行参与教师研究项目所需的各种核磁共振实验。这些实验包括：同核二维实验，如NOESY和ECOSY，由Brian Reid的小组用于研究核酸溶液结构；异核多维实验，如HMQC-NOESY，由Rachel Klevit的小组用于研究蛋白质、蛋白质-蛋白质和蛋白质-核酸复合物的结构；固体核磁共振技术，如氘宽线核磁共振、旋转共振（R2）和RFDR，Gary Drobny的研究组用于研究核酸和非常大的核酸-蛋白复合物的结构和动力学，Michael Heinekey的研究组用于研究无机金属氢化物复合物的动力学，Heinz Floss的研究组用于研究天然产物和代谢物的结构。对于使用核磁共振作为探测分子结构的主要工具的Klevit、Drobny和Reid小组来说，对更高场的需求主要是由对更高灵敏度的要求驱动的。贯穿这三个项目的一个共同主题是对研究核酸和蛋白质复合物结构的兴趣。然而，蛋白质-核酸复合物是出了名的不溶性，因此需要更高的灵敏度来研究溶液中非常低浓度的复合物。固体核磁共振实验已经被Drobny小组用来测量标记核酸的核间距离，使得研究与蛋白质非常大的复合物中的DNA和RNA结构成为可能，但是许多重要的固体核磁共振实验，即多个量子实验，其灵敏度太低，无法实际用于生物聚合物的大型复合物。我们估计，大约2.8的灵敏度提高因子将极大地扩展我们可以使用这种方法研究的分子大小领域。此外，许多其他教师将找到750兆赫兹。光谱仪对他们的研究项目有用或至关重要。这些教员包括：研究蛋白质折叠的David Baker，他需要高灵敏度的核磁共振仪器，因为折叠中间体非常不溶。研究生物材料的Pat Stayton需要高灵敏度的NMR设备来研究分子量为13000的蛋白质Streptavidin的溶液结构。Streptavidin是生物传感器的重要组成部分，在溶液中四聚体。计划研究高分子量DNA溶液动力学的J. Michael Schurr也需要高场核磁共振仪器。华盛顿大学有资格承担高场核磁共振仪器的开发工作。自1985年以来，已经设计和建造了三个核磁共振光谱仪，包括：两个500 MHz。控制台（都能够执行高分辨率和固态核磁共振实验），和一个400兆赫。固态核磁共振光谱仪（主要用于高速魔角样品旋转实验）。此外，uw最近还从Battelle的太平洋西北实验室获得了一份合同，设计、原型和建造一个900-1000 MHz的天线。美国能源部资助的环境与分子科学实验室的核磁共振。本设计工作频率为900- 1000mhz。核磁共振控制台已经在进行中，可以很容易地适应750兆赫。控制台。最后，计划与Resonance Research Inc.合作，旨在应用矩阵垫片技术产生用于多维核磁共振实验和微成像的脉冲场梯度。在这项计划的第一阶段，我们将尝试使用矩阵技术在11.75特斯拉磁铁中产生脉冲梯度。随后将应用于更高领域的系统。

ABSTRACT Funding is requested in the amount of $700,000 toward the purchase of a 17.6 Tesla NMR magnet and toward the design and construction of an accompanying NMR console. The 750 MHz. NMR console that we propose to construct, will be capable of performing the wide variety of NMR experiments required by the research programs of the participating faculty. These experiments include: homonuclear two dimensional experiments like NOESY and ECOSY, used by Brian Reid's group to study nucleic acids solution structure, heteronuclear multi-dimensional experiments like HMQC-NOESY, used by Rachel Klevit's group to study the structure of proteins, protein-protein and protein-nucleic acids complexes, solid state NMR techniques like deuterium wide line NMR, Rotational Resonance (R2) and RFDR, used by Gary Drobny's group to study the structure and dynamics of nucleic acids and of very large nucleic acid-protein complexes, and high resolution tritium NMR, used by Michael Heinekey's group to study the kinetics of inorganic metal hydride complexes and by Heinz Floss' group to study the structure of natural products and metabolites. For the groups of Klevit, Drobny, and Reid, which use NMR as a primary tool for probing molecular structure, the need for higher field is driven mainly by a requirement for higher sensitivity. A common theme running through all three programs is the interest in studying the structure of nucleic acids and proteins in complexes. However, protein-nucleic acid complexes are notoriously insoluble, hence the need for higher sensitivity to study very low concentrations of complexes in solution. Solid state NMR experiments have been used by the Drobny group to measure internuclear distances in labeled nucleic acids, making possible the study of DNA and RNA structure in very large complexes with proteins, but a number of important solid state NMR experiments, i.e. multiple quantum experiments, are simply too low in s ensitivity to be practical for use in large complexes of biopolymers. We estimate that the factor of roughly 2.8 improvement in sensitivity will greatly expand the domain of molecular size that we can study using such methods. In addition, a number of other faculty will find a 750 MHz. spectrometer useful or crucial for their research programs. These faculty include: David Baker, who studies protein folding, needs high sensitivity NMR instrumentation because folding intermediates are very insoluble. Pat Stayton, who studies bio-materials, requires high sensitivity NMR equipment to study the solution structure of the 13000 molecular weight protein Streptavidin, which is an important component in biosensors, and which tetramerizes in solution. J. Michael Schurr, who plans to study the solution dynamics of high molecular weight DNA, also requires high field NMR instrumentation. The University of Washington is qualified to carry of the development of high field NMR instrumentation. Three NMR spectrometers have been designed and built since 1985 including: two 500 MHz. consoles (both capable of performing high resolution and solid state NMR experiments), and a 400 MHz. solid state NMR spectrometer (used primarily for high speed magic angle sample spinning experiments). In addition, the U.W. has recently received a contract from Battelle's Pacific Northwest Lab to design, prototype, and construct a 900-1000 MHz. NMR for the DOE-funded Environmental and Molecular Sciences Laboratory. The design work for the 900-1000 MHz. NMR console is already in progress, and can be easily adapted to a 750 MHz. console. Finally, a collaborative effort is planned with Resonance Research Inc., aimed at applying matrix shim technology to produce pulsed field gradients for multi-dimensional NMR experiments and micro-imaging. In the first phase of this planned effort, we will attempt to use matrix technology to produce pulsed gradients in an 11.75 Tesla magnet. Applications to higher field systems will follow.