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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

用于固态 NMR 中蛋白质的可靠的带梯度 (PFG) 的转角旋转 (SAS) 探针抽象的固态核磁共振 (ssNMR) 生物技术正在成为高分辨率的首选方法整合膜蛋白（IMP）的结构测定。 ssNMR 提供了一个独特的平台研究各种生物分子超分子的蛋白质动力学和功能研究组件。虽然存在一套魔角旋转（MAS）和定向样品（OS）固态用于中小型蛋白质结构表征的核磁共振实验，这些方法面临较大系统中的几个挑战。挑战的核心是核磁共振灵敏度和分辨率。快速MAS 和1H检测实验提高了灵敏度，但受样本量限制，相对较差小各向同性化学位移色散的分辨率。此外，MAS 实验的效率很大程度上取决于通过键合和通过空间的耦合常数、溶剂抑制和相干性转子同步多脉冲应用期间的路径选择。他们还遭受敏感性丧失的困扰由于蛋白质的局部和整体运动。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核偶极耦合来提高分辨率，但效果有限仅用于稀释自旋和低伽玛 15N 检测。人们很早就认识到MAS和OS的统一已经能够将光谱应用范围扩大到大型球状蛋白和膜蛋白。切换角度旋转 (SAS) 探头将 MAS、动态角度旋转 (DAS) 和可变角度结合在一起 ssNMR 中的旋转 (VAS) 技术，并可能将各向同性和各向异性位移/耦合关联起来不止一个傅里叶维。如此强大的技术距离实际应用还很远，因为 SAS 过去的探头由于硬件故障（例如，探头的生存）而缺乏可靠性。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，快速重新定向和精确的角度再现性。技术难点和工程挑战迄今为止，探测技术仅限于两个频道。该提案寻求第二阶段资金，以继续开发可靠的转换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。第一阶段探针展示了 1H、13C 和 13C 固定调谐频率的可行性。 11.7 T 下的 15N 原子核，用于生物应用。二期探针将通过扩展技术来推进技术两个版本的调谐功能，具有两个宽带低频通道的 H/X/Y SAS-PFG 探头，和 1H/19F/X SAS-PFG 探头。此外，这些三通道探头将与市售三轴梯度线圈，以实现梯度增强光谱方法，扩散核磁共振和固态显微成像功能。这种探测器的出现将增强我们的能够开发蛋白质核磁共振研究和治疗药物筛选的新方法。