Expanding the Scope of NMR Sample Preparation

扩大 NMR 样品制备的范围

基本信息

批准号：
10089600
负责人：
Katherine Anne Henzler-Wildman
金额：
$ 20.64万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
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/10089600
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 Reproducibility Resolution Resources Risk Sampling Scientist Series Solid Specific qualifier value Structure Techniques Technology Time Titrations Training Tube Work biophysical properties biophysical techniques computerized tools cost cryogenics data acquisition design experimental study instrument macromolecule magnetic field milligram nanodisk pH gradient protein function sensor small molecule solid state solid state nuclear magnetic resonance solute structural biology success synergism technology research and development tool voltage wasting

项目摘要

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.

TR＆D 1摘要该技术研发项目针对固态NMR和结构生物学。首先，示例处理代表固态NMR的主要瓶颈，成本为昂贵的时间，钱，浪费了样本。强大的新固态NMR放松音量和标签要求，但只有当宝贵的生物样品有效地转移到微小的MAS转子中时。我们将开发工具来使此过程更快，更高效和可靠。我们还将开发更改样本条件的工具 MAS转子在实验过程中更好地评估样品的功能状态。第二，膜蛋白质在存在电压，离子，离子，离子梯度的情况下在不对称环境中的功能和pH，但是在没有这些梯度的情况下，进行了膜蛋白的结构生物学。这很难将结构与功能相关联，并创建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膜蛋白进行NMR研究的技术在跨膜电压存在下的脂质双层中，使用几种不同的pH和离子梯度在NMR数据收集过程中确保梯度稳定性的方法。我们还将开发NMR阅读在NMR数据采集过程中测量膜电位以及pH和离子梯度的传感器。这技术将直接测量膜蛋白的结构和动力学梯度的存在，揭示了跨膜梯度如何调节和驱动膜蛋白功能。由于很大一部分药物靶标是膜蛋白，它们会产生，转化，消散或该技术受跨膜梯度的调节，将产生很高的生物医学影响。