A Reliable Switched Angle Spinning (SAS) Probe with Gradients (PFG) for Proteins in Solid-State NMR

用于固态 NMR 中蛋白质的可靠的带梯度 (PFG) 的转角旋转 (SAS) 探针

• 批准号: 10667507
• 负责人: Francis DAVID Doty
• 金额: $ 64.14万
• 依托单位: DOTY SCIENTIFIC, INC.
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10667507
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A Reliable Switched Angle Spinning (SAS) Probe with Gradients (PFG) for Proteins in Solid-State NMR Abstract Solid-state NMR (ssNMR) biotechnology is emerging as a method of choice for high-resolution structure determination for integral membrane proteins (IMPs). ssNMR provides a unique platform to investigate protein dynamics and functional studies of a wide range of biomolecules in their supramolecular assemblies. While there exists a suite of magic angle spinning (MAS) and oriented sample (OS) solid state NMR experiments for structural characterization of small- and medium-sized proteins, these methods face several challenges in larger systems. Central to the challenges are NMR sensitivity and resolution. Fast MAS and 1H detected experiments improve sensitivity but are limited by sample volume and relatively poor resolution over small isotropic chemical shift dispersion. Additionally, the efficiency of MAS experiments depends largely on through-bond and through-space coupling constants, solvent suppression, and coherence pathways selection during rotor synchronized multi-pulse applications. They also suffer from sensitivity loss due to local and global motions in proteins. On the other hand, static OS NMR experiments in membrane proteins improve resolution by measuring anisotropic shifts and heteronuclear dipolar couplings but are limited to dilute spins and low gamma 15N detection only. It has long been realized that unification of MAS and OS has the ability to widen the spectroscopic applications to large globular and membrane proteins. Switched angle spinning (SAS) probes unify MAS, dynamic angle spinning (DAS) and variable angle spinning (VAS) techniques in ssNMR, and potentially correlate isotropic and anisotropic shifts/couplings in more than one Fourier dimension. Such powerful techniques are still far from practical use, because SAS probes in the past have suffered from the lack of reliability due to hardware failures such as the survival of multi-channel rf-leads, rf coil performance including B1 field strength and homogeneity, spinning stability, and lastly rapid reorientation and accurate angle reproducibility. Technical difficulties and engineering challenges thus far have limited the probe technology to only two frequency channels. This proposal seeks Phase-II funding for the continued development of a reliable switched angle spinning probe devoid of previously encountered hardware related issues and compatible with high power pulsed-field gradients. The Phase-I probe demonstrated feasibility with fixed tuning frequencies for 1H, 13C, and 15N nuclei at 11.7 T for biological applications. The phase-II probe will advance the technology by extending the tuning capabilities in two versions, an H/X/Y SAS-PFG probe with two broad-band low-frequency channels, and a 1H/19F/X SAS-PFG probe. Additionally, these triple-channel probes will be compatible with a commercially available three-axis gradient coil in order to enable gradient enhanced spectroscopic methods, diffusion NMR, and micro-imaging capabilities in solid state. The advent of such a probe will enhance our ability to develop novel methods for NMR study of proteins and screening of therapeutic drugs.

一种用于固体核磁共振蛋白质分析的高可靠性开关角自旋探针 摘要 固态NMR（ssNMR）生物技术正在成为高分辨率NMR的选择方法。 整合膜蛋白（IMP）的结构测定。ssNMR提供了一个独特的平台， 研究蛋白质动力学和各种生物分子超分子功能研究 组件.虽然存在一套魔角旋转（MAS）和定向样品（OS）固态 核磁共振实验的结构表征的小型和中型蛋白质，这些方法面临 在更大的系统中的几个挑战。挑战的核心是NMR灵敏度和分辨率。快速MAS 和1H检测实验提高了灵敏度，但受样品体积的限制， 分辨率优于小的各向同性化学位移色散。此外，MAS实验的效率 很大程度上取决于通过键和通过空间耦合常数，溶剂抑制，和相干性 转子同步多脉冲应用期间的路径选择。他们也遭受灵敏度损失 由于蛋白质的局部和全局运动。另一方面，膜中的静态OS NMR实验 蛋白质通过测量各向异性位移和异质偶极耦合来提高分辨率， 以稀释自旋和低伽马15 N探测。人们早就认识到，MAS与OS的统一， 将光谱应用扩大到大的球状和膜蛋白的能力。 开关角旋转（SAS）探头将MAS、动态角旋转（DAS）和可变角旋转（VAS）结合在一起 自旋（VAS）技术，并潜在地关联各向同性和各向异性位移/耦合， 多于一个傅立叶维度。这种强大的技术离实际应用还很远，因为SAS 过去的探测器由于硬件故障而缺乏可靠性 多通道射频导线、射频线圈性能（包括B1场强和均匀性）、旋转稳定性，以及 最后是快速重定向和精确的角度再现性。技术难题和工程挑战 到目前为止，探测技术仅限于两个频率信道。 该提案寻求第二阶段资金，用于继续开发可靠的开关角 旋转探头没有以前遇到的硬件相关问题，并与高功率兼容 脉冲场梯度第一阶段的探针证明了固定调谐频率的1H，13 C， 11.7T下的15 N核用于生物学应用。第二阶段的探测器将通过扩展 两种版本的调谐能力，一个H/X/Y SAS-PFG探头，带有两个宽带低频通道， 和1H/19 F/X SAS-PFG探针。此外，这些三通道探头将与 商业上可获得的三轴梯度线圈为了实现梯度增强光谱方法， 扩散NMR和固态显微成像能力。这种探测器的出现将增强我们的 有能力开发用于蛋白质NMR研究和治疗药物筛选的新方法。