A SAS NMR Probe for Structure Elucidation of Proteins

描述(由申请人提供)：主要基于X射线结晶学和核磁共振(核磁共振)溶液方法确定分子结构的现有方法具有局限性。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或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。核磁共振工具最近增加的一个更有前景的功能是在部分对准的解决方案中使用来自剩余偶极耦合的长程约束。通过使用双分子束的稀液晶溶液(由于其磁各向异性)获得了部分大分子排列，但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磁体的样品以魔角(相对于B/0为54.7/0)旋转时，它们与Bo的相互作用消失，它们的取向变得无序。对于角度小于54.7/0的采样旋转，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。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的磁场中以高分辨率(~0.01ppm)的高分辨率(~0.01ppm)检测多核三共振魔角旋转(MAS)；此外，需要快速(&lt；30ms)重新定位旋转轴，而不会对任何通道的旋转稳定性或射频调谐产生不利影响；此外，还具有许多额外要求，包括应用脉冲磁场梯度、稳定的温度控制、最小化1H背景信号以及与窄口径高场磁体兼容。我们以前已经在大口径磁体中实施了SAS，在低场下具有上述一些特征。初步设计评估表明，在窄孔磁体的约束下，高场SAS探头的要求是可以实现的。该第一阶段SBIR项目预计将展示适用于600 MHz窄孔磁体的高性能1H/X/YPFG-HR-SAS探头的可行性。原型探测器将由美国国立卫生研究院的Ad Bax博士进行测试。第二阶段将完成与布鲁克和瓦里安窄径光谱仪兼容的商业产品的必要开发，至少在800兆赫范围内。

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.