A SAS NMR Probe for Structure and Function Elucidation of Proteins

描述（由申请人提供）：主要基于X射线晶体学和核磁共振（NMR）溶液法的生物分子结构测定的可用方法具有局限性。虽然X射线方法快了几个数量级，但需要NMR技术来获得动态信息或生物分子在其天然环境中的信息，这两者对于功能阐明都是必不可少的。最近加入NMR工具库的更有前途的工具之一是使用部分对齐解决方案中的残余偶极耦合的长程约束。通过使用被称为双胞的盘形颗粒的稀液晶溶液已经获得了部分大分子排列，但是这不允许对排列进行所需的动态控制。几个最重要的NMR研究小组最近的分析和实验表明，新的开关角旋转（SAS）技术应该提供所需的动态控制的bicelle（因此，蛋白质）对齐。当含有负磁各向异性的盘状双胞的样品在魔角旋转（MAS）中相对于B 0以54.7倍旋转时，它们与B 0的相互作用消失，并且它们的取向变得随机。对于以小于54.7x的角度旋转的示例，它们将与垂直于旋转轴的法线对齐，而以更大的角度旋转将导致其法线与旋转轴对齐。对旋转轴的动态控制预计将提供更有效地利用残余偶极耦合中固有的键角信息所需的蛋白质排列控制。该仪器还将促进研究不溶性蛋白质所需的NMR技术，以及一种密切相关的变体，具有高性能魔角梯度的H/X/Y HR MAS，将使Frydman的超快多维NMR技术在非均匀系统中成为可能。 适用于蛋白质结构测定的SAS NMR的仪器要求极具挑战性。NMR探针必须能够在高达19 T的磁场下进行具有高分辨率（0.005 ppm）的高灵敏度间接（1H）检测的多核三重共振MAS。此外，需要快速（25 ms）旋转轴的重新定向，而不会对旋转稳定性或射频调谐产生不利影响;还有许多额外的要求，包括脉冲场梯度，稳定的温度控制，低1H背景信号，以及与窄孔（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，但仍存在许多优化和产品改进问题。第二阶段将完成与布鲁克、JEOL和Varian NB光谱仪兼容的商用产品在800 MHz频段的必要开发。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.