Acquisition of a 750 MHz NMR Spectrometer/Cryoprobe System and High Field NMR Console/Probe Upgrades for Biomolecular NMR Spectroscopy

采购 750 MHz NMR 波谱仪/冷冻探针系统以及用于生物分子 NMR 波谱学的高场 NMR 控制台/探针升级

• 批准号: 0216077
• 负责人: James Stivers
• 金额: $ 117.8万
• 依托单位: Johns Hopkins University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-15 至 2005-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0216077&HistoricalAwards=false
• 关键词: Acquisition; 750; MHz; NMR; Spectrometer

PROJECT SUMMARYA grant has been awarded to Dr. James T. Stivers at Johns Hopkins University School of Medicine to purchase a Varian 750 MHz NMR spectrometer equipped with a triple resonance probe, to upgrade existing 500 MHz and 600 MHz instruments with present generation consoles, and to purchase an additional high sensitivity triple resonance probe with a tunable X channel for the existing 500 MHz instrument. . These spectrometers will be located at the NMR facility, and will be used to significantly advance fundamental research into the structure, mechanism and dynamics of biological macromolecules using multi-dimensional heteronuclear NMR methods. This acquisition will allow the research programs of six primary NMR investigators to take advantage of revolutionary magnet and probe technologies that are essential for studying large biological molecules under dilute conditions. The 750 MHz spectrometer with cryoprobe will provide a significant increase in chemical shift dispersion and sensitivity that is essential for studying denatured states of proteins, aliphatic proton regions of nucleic acids, and obtaining spectral resolution of native 20-40 kDa proteins and large protein and protein-DNA complexes that are now under study. The 750 MHz spectrometer/cryoprobe system will allow NMR data to be acquired with sensitivity that is over 4-fold greater than the spectrometers currently being used, and will enhance the measurement of residual dipolar couplings in macromolecules. This technology will also allow the study of biological molecules that are currently inaccessible due to inherent weakness of the NMR signals, low solubility, low sample availability, or short-lived stability. The types of NMR experiments that will be enhanced using the new NMR systems will include the complete array of double and triple resonance heteronuclear experiments using isotopically 13C, 15N and/or 2H labeled protein and nucleic acid samples. In addition, the 750 MHz spectrometer will be used for homonuclear 1H experiments, such as DQF-COSY, TOCSY and NOESY on macromolecules that may be difficult to isotope label, or in situations where resolution is critical, such as the heavily overlapped sugar proton regions of nucleic acids, or the aromatic regions of proteins. The NMR experiments will be used to obtain chemical shift assignments, measure structural restraints for macromolecular samples in the form of NOE derived distances, J-coupling derived dihedral angles, and using residual dipolar couplings derived atomic vectors and alignment tensors. Additionally, heteronuclear NMR experiments will be used to study both fast (ps) and slower (ms to ms) dynamics in macromolecular systems. These instruments will be used to facilitate NMR studies on biological macromolecules and their complexes in efforts to probe the physical and chemical basis for protein and nucleic acid function. We anticipate that these fundamental discoveries will impact protein engineering efforts, protein fold predictions, as well as our ability to target enzymes with small molecule inhibitors and activators. The upgraded NMR facility will enhance student training by providing a user friendly instrument, and ample user time that is required for student researchers to become comfortable in the execution of modern NMR experiments. The participating investigators will initiate a new course covering the basics of executing homonuclear and heteronuclear NMR experiments on these new instruments. This course will augment the existing graduate curriculum, and will attract advanced undergraduate students from local universities, including minority serving inttitutuons.

项目概述授予约翰霍普金斯大学医学院的James T. Stivers博士，用于购买配备三重共振探头的瓦里安750 MHz核磁共振光谱仪，升级现有的500 MHz和600 MHz仪器与当前一代控制台，并为现有的500 MHz仪器购买额外的高灵敏度三重共振探头，带有可调谐的X通道。这些光谱仪将位于核磁共振设施，并将用于使用多维异核核磁共振方法显著推进生物大分子结构，机制和动力学的基础研究。此次收购将使六个主要核磁共振研究人员的研究项目能够利用革命性的磁铁和探针技术，这些技术对于在稀释条件下研究大型生物分子至关重要。配备冷冻探针的750 MHz光谱仪将显著提高化学位移色散和灵敏度，这对于研究蛋白质的变性状态、核酸的脂肪族质子区域以及获得天然20-40 kDa蛋白质和大蛋白质以及蛋白质- dna复合物的光谱分辨率至关重要。750 MHz光谱仪/低温探针系统将允许以比目前使用的光谱仪高4倍以上的灵敏度获取NMR数据，并将增强对大分子中残余偶极耦合的测量。这项技术还将允许研究目前由于核磁共振信号固有的弱点、低溶解度、低样品可用性或短暂稳定性而无法进入的生物分子。使用新的核磁共振系统将增强核磁共振实验的类型，包括使用同位素13C、15N和/或2H标记的蛋白质和核酸样品进行完整的双共振和三共振异核实验。此外，750 MHz光谱仪将用于可能难以同位素标记的大分子的同核1H实验，如DQF-COSY， TOCSY和noesi，或在分辨率至关重要的情况下，如核酸的重度重叠糖质子区，或蛋白质的芳香区。核磁共振实验将用于获得化学位移分配，以NOE衍生距离、j耦合衍生二面角的形式测量大分子样品的结构约束，并使用剩余偶极耦合衍生的原子矢量和对准张量。此外，异核磁共振实验将用于研究大分子体系中的快速（ps）和慢速（ms - to - ms）动力学。这些仪器将用于促进生物大分子及其复合物的核磁共振研究，以探索蛋白质和核酸功能的物理和化学基础。我们预计这些基础发现将影响蛋白质工程的努力，蛋白质折叠预测，以及我们用小分子抑制剂和激活剂靶向酶的能力。升级后的核磁共振设备将通过提供用户友好的仪器来加强学生的培训，并为学生研究人员提供充足的用户时间，以适应现代核磁共振实验的执行。参与的研究人员将启动一门新的课程，涵盖在这些新仪器上执行同核和异核NMR实验的基础知识。本课程将补充现有的研究生课程，并将吸引本地大学（包括少数民族院校）的高级本科生。