Expanding the Scope of NMR Sample Preparation

扩大 NMR 样品制备的范围

• 批准号: 10573324
• 负责人: Katherine Anne Henzler-Wildman
• 金额: $ 18.74万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-02 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10573324
• 关键词: Activities of Daily Living; Aphorisms; Benchmarking; Biological; Cell Membrane Permeability; Cells; Characteristics; Chemicals; Data Collection; Disease; Drug Targeting; Ensure; Environment; Formulation; Goals; Hour; Hydration status; Individual; Integral Membrane Protein; Investments; Ionic Strengths; Ions; Isotope Labeling; Knowledge; Label; Ligands; Light; Lipid Bilayers; Lipids; Liposomes; Magic; Measurement; Measures; Membrane; Membrane Potentials; Membrane Proteins; Methods; Micelles; Molecular Weight; Nuclear; Persons; Pharmaceutical Preparations; Phase; Physiologic pulse; Physiological; Preparation; Process; Proton-Motive Force; Recycling; Relaxation; Reproducibility; Resolution; Resources; Sampling; Scientist; Series; Solid; Specific qualifier value; Structure; Techniques; Technology; Time; Titrations; Training; Tube; Viscosity; Work; biophysical properties; biophysical techniques; computerized tools; cost; cryogenics; data acquisition; design; experimental study; instrument; macromolecule; magnetic field; milligram; nanodisk; pH gradient; protein function; risk mitigation; sensor; small molecule; solid state; solid state nuclear magnetic resonance; solute; structural biology; success; synergism; technology research and development; tool; voltage; wasting

TR&D 1 SUMMARY This technology research and development project targets two major challenges for solid-state NMR and structural biology. First, sample handling represents a major bottleneck for solid-state NMR, costing time, money, and wasted sample. Powerful new solid-state NMR relax the volume and labeling requirements, but only if precious biological samples can be efficiently transferred into tiny MAS rotors. We will develop tools to make this process faster, more efficient and reliable. We will also develop tools to change sample conditions in the MAS rotor to better assess the functional state of a sample during experiments. Second, membrane proteins function in an asymmetric environment in the presence of transmembrane gradients of voltage, ions, and pH, but structural biology of membrane proteins is carried out in the absence of these gradients. This makes it difficult to correlate structure with function and creates one of the largest current knowledge gaps in structural biology. The project develops technology to perform NMR studies of membrane proteins embedded in lipid bilayers in the presence of transmembrane voltage, pH and ion gradients using several different approaches to ensure gradient stability during NMR data collection. We will also develop NMR-readout sensors to measure the membrane potential and pH and ion gradients during NMR data acquisition. This technology will enable direct measurement of the structure and dynamics of membrane proteins in the presence of gradients, revealing how transmembrane gradients regulate and drive membrane protein function. Since a large percentage of drug targets are membrane proteins that create, transform, dissipate or are regulated by transmembrane gradients, this technology will have high biomedical impact.

研发 1 总结 该技术研发项目针对固态核磁共振和核磁共振的两大挑战 结构生物学。首先，样品处理是固态 NMR 的主要瓶颈，耗费时间， 钱，浪费了样品。强大的新型固态 NMR 放宽了体积和标签要求，但 只有当珍贵的生物样本能够有效地转移到微小的 MAS 转子中时。我们将开发工具 使这个过程更快、更高效、更可靠。我们还将开发工具来改变样品条件 MAS 转子可在实验过程中更好地评估样品的功能状态。二、膜 蛋白质在存在电压、离子、跨膜梯度的不对称环境中发挥作用 和 pH 值，但膜蛋白的结构生物学是在没有这些梯度的情况下进行的。这 使得很难将结构与功能联系起来，并造成了当前最大的知识差距之一 结构生物学。该项目开发了对嵌入的膜蛋白进行核磁共振研究的技术 在存在跨膜电压、pH 和离子梯度的情况下，使用几种不同的方法在脂质双层中 确保 NMR 数据收集过程中梯度稳定性的方法。我们还将开发核磁共振读数仪 传感器在 NMR 数据采集过程中测量膜电位、pH 值和离子梯度。这 技术将能够直接测量膜蛋白的结构和动力学 梯度的存在，揭示跨膜梯度如何调节和驱动膜蛋白功能。 由于大部分药物靶标是膜蛋白，它们产生、转化、消散或 该技术受跨膜梯度调节，将产生巨大的生物医学影响。