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: 10456218
• 负责人: Francis DAVID Doty
• 金额: $ 66.84万
• 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/10456218
• 关键词: 2,4-Dinitrophenol; 2019-nCoV; 3-Dimensional; Academia; Alzheimer' s Disease; Amyloid beta-Protein; Area; Biological; Biological Process; Biophysics; Biotechnology; Businesses; Cell Nucleus; Cell surface; Chemicals; Communities; Complement; Complex; Coupled; Coupling; Crystallization; Detection; Development; Device or Instrument Development; Diffusion; Dimensions; Disease; Drug Binding Site; E protein; Elements; Engineering; Environment; Face; Failure; Frequencies; Funding; Goals; Health; Industry; Integral Membrane Protein; Laboratories; Magic; Magnetic Resonance Imaging; Measures; Mechanics; Membrane; Membrane Proteins; Methods; Modeling; Motion; Motor; Pathway interactions; Peptide Hydrolases; Peptides; Performance; Pharmaceutical Preparations; Phase; Phospholipids; Physiologic pulse; Physiological; Play; Preparation; Process; Protein Dynamics; Proteins; RF coil; Reproducibility; Research; Research Personnel; Research Proposals; Resolution; Roentgen Rays; Role; Sampling; Solid; Solvents; Speed; Structure; System; Techniques; Technology; Temperature; Tertiary Protein Structure; Therapeutic; Time; Transmembrane Domain; biophysical properties; drug candidate; drug development; drug discovery; drug market; experimental study; flexibility; globular protein; imaging capabilities; improved; instrument; interest; macromolecule; novel; operation; protein aggregation; protein protein interaction; screening; solid state; solid state nuclear magnetic resonance; structural biology; trend; virtual

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.

一种可靠的固体核磁共振蛋白质梯度开关角度自旋(SAS)探针 摘要 固态核磁共振(SsNMR)生物技术正在成为一种高分辨率的选择方法 完整膜蛋白(IMPS)的结构测定。SS核磁共振提供了一个独特的平台来 超分子生物大分子的蛋白质动力学和功能研究 装配。虽然存在一套魔角旋转(MAS)和定向样品(OS)固态 用核磁共振实验表征中小型蛋白质的结构，这些方法面临 更大系统中的几个挑战。这些挑战的核心是核磁共振的灵敏度和分辨率。FAST MAS 1H检测实验提高了灵敏度，但受样本量的限制，相对较差 小的各向同性化学位移弥散的分辨率。此外，MAS实验的效率 在很大程度上取决于贯通键和贯通空间耦合常数、溶剂抑制和连贯性 转子同步多脉冲应用中的路径选择。他们还遭受着敏感度丧失的痛苦。 由于蛋白质的局部和整体运动。另一方面，膜中的静态OS核磁共振实验 蛋白质通过测量各向异性位移和异核偶极偶联来提高分辨率，但受到限制 仅用于稀释自旋和低伽马15N探测。人们早就意识到MAS和OS的统一已经 将光谱应用范围扩大到大的球状和膜蛋白的能力。 开关角度旋转(SAS)探头统一了MAS、动态角度旋转(DAS)和可变角度 单核磁共振中的自旋(VAS)技术，以及潜在地关联各向同性和各向异性位移/耦合 不止一个傅立叶维度。如此强大的技术还远远没有实际应用，因为SAS 过去的探头由于硬件故障而缺乏可靠性，例如 多通道射频引线，射频线圈性能，包括B1场强和均匀性，旋转稳定性，以及 最后，快速重定向和准确的角度重复性。技术难题和工程挑战 到目前为止，探测技术仅限于两个频道。 这项提议寻求第二阶段的资金，以继续发展可靠的切换角度 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。I相探头证明了在1H、13C和 15N原子核，11.7T，用于生物应用。二期探测器将通过扩展技术来推进技术 两个版本的调谐能力，带有两个宽带低频通道的H/X/Y SAS-PFG探头， 1H/19F/X SAS-PFG探头。此外，这些三通道探头将与 市场上可买到的三轴梯度线圈为了实现梯度增强的光谱方法， 扩散核磁共振和固态显微成像能力。这种探测器的出现将加强我们的 具有开发蛋白质核磁共振研究和治疗药物筛选的新方法的能力。