Small and Mechanosensitive Membrane Proteins Studied with DNA-based Tools

使用基于 DNA 的工具研究小型机械敏感膜蛋白

• 批准号: 10581927
• 负责人: Thorsten Lars Schmidt
• 金额: $ 25万
• 依托单位: KENT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10581927
• 关键词: Aging; Biophysics; Buffers; Collaborations; Cryoelectron Microscopy; DNA; Development; Funding; Grant; Image; Individual; Laboratories; Lipids; Membrane Proteins; Methods; Modernization; Noise; Parents; Phase; Preparation; Process; Research; Resolution; Sampling; Scanning Probe Microscopes; Signal Transduction; Stains; Structure; Time; Vacuum; Wait Time; Work; aged; base; imaging modality; instrument; nanodisk; novel; particle; protein structure; repaired; single molecule; tool

Project Summary I am requesting funds to purchase an Atomic Force Microscope (AFM) to replace an aged and failing AFM that requires frequent major repairs. AFM is a vital method for the work proposed in the parent R35 grant which will provide new DNA-based tools that will overcome several of the challenges associated with MP structure deter- mination by single-molecule cryo EM. An AFM is needed for the development phase of our DNA-lipid nanodiscs, as AFMs produce images of individual particles with a higher resolution, contrast and signal to noise ratio than other imaging methods and no other method provides topographical information. Instant access to this instrument in our laboratory avoids long wait times thus shortening the cycles of iterative improvements of our DNA-based tools. AFM sample preparation is also much faster and requires less material than TEM grid sample preparations while avoiding staining, drying and transfer into a vacuum. In AFM, samples are imaged in native buffers, instead. The new instrument supports imaging with video frame rates which would allow us to study dynamic processes including nanodisc fusion and lipid exchange. Moreover, the accelerated imaging dramatically reduces the time that is required to image a statistically relevant number of particles which is needed for a biophysical quantification of structural parameters and synthesis yields of the DNA rings. Finally, the requested instrument will be the first modern AFM on the campus and I therefore expect interesting collaboration requests that will leverage our expertise in AFM.

项目摘要 我要求资金购买原子力显微镜（AFM），以取代老化和失败的AFM， 需要经常大修。原子力显微镜是一个重要的方法，在父R35赠款，将 提供新的基于DNA的工具，将克服与MP结构相关的几个挑战， 通过单分子冷冻EM测定。 我们的DNA-脂质纳米盘的开发阶段需要AFM，因为AFM产生个体的图像。 具有比其他成像方法更高的分辨率、对比度和信噪比的粒子， 方法提供地形信息。在我们的实验室中立即使用该仪器避免了长时间的等待 从而缩短了我们基于DNA的工具的迭代改进周期。AFM样品制备 也比TEM网格样品制备快得多，需要的材料更少，同时避免了染色、干燥 并转移到真空中。在AFM中，样品在天然缓冲液中成像。新仪器支持 用视频帧速率成像，这将使我们能够研究动态过程，包括纳米椎间盘融合， 脂质交换此外，加速的成像显著地减少了成像所需的时间。 结构参数的生物物理定量所需的统计学相关颗粒数量 和DNA环的合成产率。最后，要求的仪器将是第一个现代原子力显微镜上的 校园，因此，我期待有趣的合作请求，将利用我们在AFM的专业知识。