A SAS NMR Probe for Structure 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）溶液法的分子结构测定可用方法具有局限性。虽然X射线方法快几个数量级，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。最近增加的NMR工具中比较有前途的一个是使用部分对齐的解决方案中的残余偶极耦合的长程约束。通过使用双胞的稀液晶溶液（由于它们的磁各向异性）已经获得了部分大分子排列，但是这不允许对部分排列的量和方向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以魔角（相对于B/0为54.7/0）旋转时，它们与Bo的相互作用消失，并且它们的取向变得随机。对于以小于54.7/0的角度旋转的示例，它们会与垂直于旋转轴的法线对齐，而以更大的角度旋转会导致它们的法线与旋转轴对齐。对自旋轴的动态控制预计将提供更有效地利用剩余偶极耦合中固有的键角信息所需的蛋白质对齐控制。 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的磁场下进行多核三共振魔角旋转（MAS），并具有高灵敏度间接（1H）检测和高分辨率（~0.01 ppm）。此外，需要快速（<30 ms）重新定向旋转轴，而不会对任何通道上的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，包括应用脉冲场梯度，稳定的温度控制，最小化1H背景信号，以及与窄孔高场磁体的兼容性。我们以前实施了SAS在宽口径磁铁与一些上述功能在低场。初步设计评估表明，高场SAS探头的要求，可以实现窄孔磁铁的约束内。该第一阶段SBIR项目预计将证明适用于600 MHz窄孔磁体的高性能1H/X/Y PFG-HR-SAS探头的可行性。探针原型将由NIH的Ad Bax博士进行测试。第二阶段将完成与布鲁克和瓦里安窄口径光谱仪兼容的商业产品所需的开发，其视场至少可达800 MHz。

DESCRIPTION (provided by applicant): Available methods for 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 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 bicelles (owing to their magnetic anisotropy), but this does not permit the needed dynamical control over the amount and direction of the partial alignment. Very recent analysis and experiments by several foremost NMR research groups indicate novel Switched Angle Spinning (SAS) techniques should provide the needed dynamic control over the bicelle (and hence, the protein) alignment. When a sample containing discoidal bicelles of negative magnetic anisotropy is spun at the Magic Angle (54.7/0 with respect to B/0), their interaction with Bo vanishes and their orientation becomes random. For sample spinning at angles less than 54.7/0, 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. The instrumental requirements of SAS NMR suitable for protein structure determination are extremely challenging. The NMR probe must be capable of multinuclear triple-resonance magic angle spinning (MAS) with highly sensitive indirect (1H) detection with high resolution (~0.01 ppm) at fields up to 19 T. In addition, rapid (<30 ms) reorientation of the spinning axis is required without adversely affecting spinning stability or rf tuning on any channel; and there are a number of additional requirements, including the application of pulsed field gradients, stable temperature control, minimization of 1H background signals, and compatibility with narrow-bore high-field magnets. We have previously implemented SAS in wide-bore magnets with some of the above features at low fields. Preliminary design evaluation suggests the requirements of the high-field SAS probe can be realized within the constraints of the narrow bore magnet. This Phase I SBIR project is expected to demonstrate feasibility of a high-performance 1H/X/Y PFG-HR-SAS probe suitable for a narrow-bore magnet at 600 MHz. The prototype probe will be tested by Dr. Ad Bax at the NIH. The Phase II will complete the developments necessary for commercial products compatible with Bruker and Varian narrow-bore spectrometers at fields at least up to 800 MHz.