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.

可靠的开关角度旋转（SAS）探针，具有梯度（PFG），用于固态NMR中的蛋白质 抽象的 固态NMR（SSNMR）生物技术正在成为一种高分辨率的选择方法 整体膜蛋白（IMPS）的结构测定。 SSNMR提供了一个独特的平台 研究其超分子中多种生物分子的蛋白质动力学和功能研究 集会。同时存在一套魔术角旋转（MAS）和定向样品（OS）固态的套件 NMR实验用于中小型蛋白质的结构表征，这些方法面对 在较大系统中有几个挑战。挑战的核心是NMR敏感性和解决方案。快速MAS 1小时检测到的实验提高了灵敏度，但受样品体积和相对较差的限制 小型各向同性化学位移分散剂的分辨率。另外，MAS实验的效率 在很大程度上取决于循环和通过空间耦合常数，溶剂抑制和连贯性 转子同步多脉冲应用中的途径选择。他们也遭受敏感性损失 由于蛋白质的局部和全球运动。另一方面，膜上的静态OS NMR实验 蛋白质通过测量各向异性移位和异核偶极耦合来改善分辨率，但受到限制 仅稀释旋转和低γ15N检测。长期以来已经意识到MAS和OS的统一 扩大光谱应用在大型球状和膜蛋白上的能力。 开关角旋转（SAS）探针统一MAS，动态角度旋转（DAS）和可变角度 SSNMR中的旋转（VAS）技术，并可能与各向同性和各向异性移位/耦合相关联 一个以上的傅立叶维度。如此强大的技术仍然远非实际使用，因为SAS 过去的探针遭受了由于硬件故障的缺乏可靠性的困扰，例如生存 多通道RF铅，RF线圈性能，包括B1场强度和同质性，旋转稳定性和 最后，快速的重新定位和准确的角度可重复性。技术困难和工程挑战 迄今为止，将探针技术限制在两个频道。 该提案寻求II阶段的资金，以持续开发可靠的开关角度 旋转探针没有以前遇到的硬件相关问题，并且与高功率兼容 脉冲场梯度。 I期探测器在固定调谐频率的1H，13C和 15n核在11.7 t的生物应用。 II期探测器将通过扩展来推进技术 两种版本的调整功能，一个H/X/Y SAS-PFG探针，具有两个宽带低频通道， 和1H/19F/X SAS-PFG探针。此外，这些三通道探针将与 市售的三轴梯度线圈，以实现梯度增强的光谱方法， 扩散NMR和固态的微成像功能。这样的调查的出现将增强我们的 有能力开发新的NMR蛋白质研究和治疗药物筛查的能力。