A SAS NMR Probe for Structure and Function Elucidation of Proteins

描述(申请人提供)：生物分子结构测定的现有方法，主要基于X射线结晶学和核磁共振(核磁共振)溶液方法，具有局限性。虽然X射线方法的速度要快几个数量级，但核磁共振技术需要获得动力学信息，即关于其自然环境中生物分子的信息，这两者对于功能阐明都是必不可少的。核磁共振工具库最近增加的一个更有前景的功能是在部分对准的溶液中使用来自残留偶极耦合的长程约束。通过使用被称为双分子的盘状颗粒的稀液晶溶液，已经获得了部分大分子排列，但这不允许对排列进行所需的动态控制。几个最重要的核磁共振研究小组最近的分析和实验表明，新的开关角度旋转(SAS)技术应该可以提供对双细胞(因此，蛋白质)排列所需的动态控制。当含有具有负磁各向异性的盘状双极的样品在魔角旋转(MAS)中相对于B0以54.7倍的速度旋转时，它们与B0的相互作用消失，它们的取向变得无序。对于角度小于54.7倍的样本旋转，它们与垂直于旋转轴的法线对齐，而以较大角度旋转会使其法线与旋转轴对齐。对旋转轴的动态控制有望提供更有效地利用残留偶极耦合中固有的键角信息所需的蛋白质对齐控制。该仪器还将促进研究不溶性蛋白质所需的核磁共振技术，以及一个密切相关的变种，具有高性能魔角梯度的H/X/Y HR MAS，将使Frydman在非均质体系中的超快多维核磁共振技术成为可能。 适用于蛋白质结构测定的SAS核磁共振仪器要求极具挑战性。核磁共振探头必须能够在高达19T的磁场下进行高分辨率(0.005 ppm)的高分辨率(0.005 ppm)的多核三重共振MAS检测；此外，还需要快速(25ms)重定向旋转轴，而不会对旋转稳定性或RF调谐产生不利影响；此外，还具有许多额外要求，包括脉冲场梯度、稳定的温度控制、低1H背景信号以及与窄口径(NB)高场磁体的兼容性。第一阶段项目证明了适用于600 MHz Nb磁体的高性能1H/X/Y PFG-HR-SAS探头的可行性，但仍存在一些优化和产品改进问题。第二阶段将完成与布鲁克、JEOL和瓦里安Nb光谱仪兼容的商业产品所需的这些开发，磁场最高可达800 MHz。第二阶段将包括由NIH-NIDDK首席科学家Ad Bax博士对仪器进行测试。

DESCRIPTION (provided by applicant): Available methods for biological molecular structure determination, based primarily on x-ray crystallography and Nuclear Magnetic Resonance (NMR) solution methods, have limitations. While the x-ray methods are several orders of magnitude faster, NMR techniques are required to obtain dynamical information, or information on biological molecules in their native environments, both of which are essential for function elucidation. One of the more promising recent additions to the arsenal of NMR tools has been the use of long-range constraints from residual dipolar couplings in partially aligned solutions. Partial macromolecule alignment has been obtained by using dilute liquid crystal solutions of disc-shaped particles called bicelles, but this does not permit the needed dynamical control over the alignment. Very recent analyses and experiments by several foremost NMR research groups indicate novel Switched Angle Spinning (SAS) techniques should provide the needed dynamic control over the bicelle (hence, the protein) alignment. When a sample containing discoidal bicelles of negative magnetic anisotropy is spun at 54.7x with respect to B0 in Magic Angle Spinning (MAS), their interaction with B0 vanishes and their orientation becomes random. For sample spinning at angles less than 54.7x, they align with their normals perpendicular to the spinning axis, while spinning at greater angles causes their normals to align with the spinning axis. Dynamic control over the spinning axis is expected to provide the protein alignment control needed for more effective utilization of the bond angle information inherent in the residual dipolar coupling. This instrument will also facilitate NMR techniques needed for the study of insoluble proteins, and a closely related variant, an H/X/Y HR MAS with a high-performance Magic Angle Gradient, will enable Frydman's ultra-fast multidimensional NMR techniques in inhomogeneous systems. The instrumental requirements of SAS NMR suitable for protein structure determination are extremely challenging. The NMR probe must be capable of multinuclear triple-resonance MAS with highly sensitive indirect (1H) detection with high resolution (0.005 ppm) at fields up to 19 T. In addition, rapid (25 ms) reorientation of the spinning axis is required without adversely affecting spinning stability or rf tuning; and there are a number of additional requirements, including pulsed field gradients, stable temperature control, low 1H background signals, and compatibility with narrow-bore (NB) high-field magnets. The Phase I project demonstrated feasibility of a high-performance 1H/X/Y PFG-HR-SAS probe suitable for a NB magnet at 600 MHz, though a number of optimization and product refinement issues remain. The Phase II will complete these developments necessary for commercial products compatible with Bruker, JEOL, and Varian NB spectrometers at fields up to 800 MHz. The Phase II will include testing of the instruments by Dr. Ad Bax, chief scientist at NIH-NIDDK.