A SAS NMR Probe for Structure and Function Elucidation of Proteins

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)的解决方法，都有局限性。而x射线方法是几个数量级的 更快的速度，需要核磁共振技术来获得动态信息，即关于生物分子的信息 在它们的原生环境中，这两者对于功能阐明都是必不可少的。最近更有希望的一个 对核磁共振工具武器库的补充是使用来自剩余偶极耦合的长程约束 部分一致的解决方案。通过使用稀释的液晶溶液获得了部分大分子取向 DJSC形状的颗粒被称为双列，但这不允许对排列进行必要的动态控制。 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)技术应该提供对双细胞(因此，蛋白质)对齐所需的动态控制。 当含有具有负磁各向异性的盘状双磁体的样品相对于B0以54.7°的速度旋转时， 魔角旋转(MAS)，它们与B0的相互作用消失，它们的方向变得随机。样例 以小于54.7°的角度旋转时，它们与垂直于旋转轴的法线对齐，同时以 角度越大，它们的法线就会与旋转轴对齐。期望对旋转轴进行动态控制 为了提供更有效地利用键角信息所需的蛋白质排列控制， 剩余偶极耦合。该仪器还将促进研究不溶物质所需的核磁共振技术 蛋白质，以及一个密切相关的变体，具有高性能魔角梯度的H/X/Y HR MAS，将使 Frydman在非均质体系中的超快多维核磁共振技术。 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。 核磁共振探针必须能够进行具有高灵敏度的间接(1H)检测的多核三重共振MAS 高分辨率(0.005 ppm)，最高可达19 T。此外，还需要快速(25 Ms)重定向旋转轴 不会对旋转稳定性或射频调谐产生不利影响；还有一些额外要求，包括 脉冲场梯度，稳定的温度控制，低1H背景信号，并与窄孔(NB)兼容 高场磁铁。一期工程论证了高性能1H/X/Y PFG-HR-SAS探头的可行性 适用于600 MHz的Nb磁体，但仍存在一些优化和产品改进问题。这个 第二阶段将完成与Bruker、JEOL和 瓦里安Nb光谱仪，磁场高达800 MHz。第二阶段将包括由Ad Bax博士对仪器进行测试， NIH-NIDDK的首席科学家。

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 djsc-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 54.7¿ 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 54.7¿, 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.