A SAS NMR Probe for Structure and Function Elucidation of Proteins

用于阐明蛋白质结构和功能的 SAS NMR 探针

• 批准号: 6990433
• 负责人: Francis DAVID Doty
• 金额: $ 61.99万
• 依托单位: DOTY SCIENTIFIC, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-15 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6990433
• 关键词: bioengineering /biomedical engineering; biomedical equipment development; macromolecule; molecular probes; nuclear magnetic resonance spectroscopy; protein structure function; structural biology

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.

描述（由申请人提供）：现有的生物分子结构测定方法，主要基于x射线晶体学和核磁共振（NMR）溶液方法，有局限性。虽然x射线方法要快几个数量级，但需要核磁共振技术来获得动态信息或生物分子在其原生环境中的信息，这两者对于功能阐明都是必不可少的。最近在核磁共振工具库中增加的一个更有前途的工具是使用部分对准溶液中残余偶极耦合的远程约束。部分大分子排列已通过使用被称为双胞体的圆盘状颗粒的稀释液晶溶液获得，但这不允许对排列进行所需的动态控制。最近几个最重要的核磁共振研究小组的分析和实验表明，新的开关角旋转（SAS）技术应该提供所需的对小细胞（因此，蛋白质）排列的动态控制。当磁各向异性为负的盘状双胞体在魔角旋转（MAS）中以54.7倍于B0旋转时，它们与B0的相互作用消失，它们的取向变得随机。对于角度小于54.7x的样品，它们与垂直于自转轴的法线对齐，而以更大的角度旋转则使其法线与自转轴对齐。对自旋轴的动态控制有望提供所需的蛋白质定位控制，以便更有效地利用剩余偶极耦合中固有的键角信息。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体，一个具有高性能魔角梯度的H/X/Y HR MAS，将使Frydman的超快速多维核磁共振技术在非均匀系统中实现。