M. Hong RT&D

洪明 RT

• 批准号: 10224250
• 负责人: Mei Hong
• 金额: $ 5.6万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10224250
• 关键词: 3-Dimensional; Address; Amyloid Fibrils; Anisotropy; Area; Automobile Driving; Binding; Biological; Biosynthetic Proteins; Cell Wall; Chemicals; Cholesterol; Coupling; Disease; Electrons; Frequencies; Harvest; Investigation; Ion Channel; Isotope Labeling; Label; Ligands; Magnetic Resonance; Measurable; Measurement; Measures; Membrane; Membrane Proteins; Methods; Modernization; Molecular; Motion; Nature; Pharmaceutical Chemistry; Physiologic pulse; Production; Protein Dynamics; Proteins; Reagent; Relaxation; Resolution; Site; Structure; Techniques; Technology; Testing; Time; Water; base; beta pleated sheet; experimental study; magnetic field; novel; protein function; protein structure; radio frequency; small molecule; solid state nuclear magnetic resonance; structural biology; three dimensional structure

Project Summary of TR&D 2 – High-field and fast-MAS SSNMR methods for biomolecular structure and dynamics (PI: Hong) TR&D 2 seeks to develop new solid-state NMR technologies to determine protein structure and dynamics and to produce isotopically labeled cholesterol for membrane protein structural biology. Modern solid-state NMR based structure determination approaches focus on 13C and 15N based resonance assignment and distance measurements. However, the low gyromagnetic ratios of 13C and 15N limit the measurable distances to less than ~8 Å, which makes it difficult to determine protein three-dimensional folds and quaternary structures. Aim 1 of this TR&D is to overcome this bottleneck in structure determination by developing 19F-based MAS NMR techniques to measure 19F-19F, 19F-13C and 19F-1H distances, potentially to 2 nm. These experiments will be developed for high field and fast MAS conditions to achieve the high-resolution and sensitivity necessary for structure determination. The impact of 19F chemical shift anisotropy on dipolar recoupling will be investigated systematically. Aim 2 of this TR&D is to develop 13C-2H correlation NMR techniques to measure protein dynamics at high magnetic fields with higher angular sensitivity than before. This aim addresses the limitation of 13C-1H and 15N-1H dipolar-coupling based techniques, which are increasingly less sensitive to small- amplitude motions as the magnetic field strength and MAS frequency increase. We will harvest the power of the large 2H quadrupolar coupling for sensing molecular orientations by developing robust polarization transfer techniques between 2H spins and 13C or 15N spins, so that we can measure 13C and 15N resolved 2H quadrupolar spectra. In aim 3 of this TR&D, we will develop biosynthetic methods for high-yield production of 13C and 2H-labeled cholesterol for membrane protein structural biology. By partnering with four driving biomedical projects, we will test and deploy the technologies in this TR&D to address outstanding biological questions in the areas of membrane proteins and cell walls.

TR＆D 2的项目摘要 - 生物分子的高场和快速SSNMR方法 结构和动力学（PI：Hong） TR＆D 2试图开发新的固态NMR技术来确定蛋白质结构 和动力学并生产用于膜蛋白结构生物学的同位素标记的胆固醇。 现代固态NMR的结构确定方法集中于13C和15N 共振分配和距离测量值。但是，低旋转磁比为13C和 15N可测量的距离将可测量的距离限制在小于〜8Å，这使得难以确定蛋白质 三维褶皱和第四纪结构。此tr＆d的目标1是克服这个瓶颈 通过开发基于19F的MAS NMR技术来测量19F-19F，19F-13C来确定结构。 和19f-1h距离，可能为2 nm。这些实验将针对高场和快速开发 MAS条件以实现结构确定所需的高分辨率和灵敏度。 19F化学移动各向异性对偶极回合的影响将被系统地研究。 该TR＆D的目标2是开发13C-2H相关NMR技术，以测量蛋白质动力学 高磁场具有比以前更高的角度灵敏度。这个目的解决了 基于13C-1H和15N-1H偶性耦合技术，这些技术对小对小的敏感 随着磁场强度和MAS频率的增加，放大器的运动。我们将恢复 大型2H四极耦合的功率通过开发强大的 2H旋转和13C或15N旋转之间的极化转移技术，以便我们可以测量13C 和15N解析了2H四极光谱。在此TR＆D的AIM 3中，我们将开发生物合成方法 用于高收益生产13C和2H标记的胆固醇，用于膜蛋白结构生物学。经过 与四个驾驶生物医学项目合作，我们将在此TR＆D中测试和部署这些技术 解决膜蛋白和细胞壁领域中的杰出生物学问题。