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

项目摘要一笔赠款已授予詹姆斯T博士。与约翰霍普金斯大学医学院的Stivers合作，购买配备三重共振探头的Varian 750 MHz NMR光谱仪，使用当前一代控制台升级现有的500 MHz和600 MHz仪器，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X通道）。 . 这些光谱仪将位于NMR设施中，并将用于使用多维异相NMR方法显着推进对生物大分子的结构，机制和动力学的基础研究。 此次收购将使六名主要NMR研究人员的研究计划能够利用革命性的磁体和探针技术，这些技术对于在稀释条件下研究大型生物分子至关重要。 带有冷冻探针的750 MHz光谱仪将提供化学位移分散和灵敏度的显著增加，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目前正在研究的天然20-40 kDa蛋白质和大蛋白质和蛋白质-DNA复合物的光谱分辨率至关重要。 750 MHz光谱仪/冷冻探针系统将允许以比目前使用的光谱仪高4倍以上的灵敏度采集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NMR信号的固有弱点，低溶解度，低样品可用性或短期稳定性而无法获得的生物分子。 将使用新的NMR系统增强的NMR实验类型将包括使用同位素13 C，15 N和/或2 H标记的蛋白质和核酸样品的双共振和三共振异谱实验的完整阵列。 此外，750 MHz光谱仪还将用于同位素1H实验，如DQF-COSY、TOCSY和NOESY，用于难以同位素标记的大分子，或用于分辨率至关重要的情况，如核酸的糖质子区域或蛋白质的芳香区域。 NMR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，并使用残余偶极耦合衍生原子矢量和排列张量。 此外，异相NMR实验将用于研究大分子系统中的快速（ps）和慢速（ms到ms）动力学。 这些仪器将用于促进生物大分子及其复合物的NMR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，这些基本的发现将影响蛋白质工程的努力，蛋白质折叠的预测，以及我们的能力，目标酶与小分子抑制剂和激活剂。 升级后的NMR设施将通过提供用户友好的仪器和充足的用户时间来加强学生培训，这是学生研究人员在执行现代NMR实验时所需的。 参与的研究人员将开始一个新的课程，涵盖了在这些新仪器上执行同源和异源NMR实验的基础知识。 这门课程将加强现有的研究生课程，并将吸引当地大学的高级本科生，包括少数民族服务机构的学生。

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.