Small and Mechanosensitive Membrane Proteins Studied with DNA-based Tools

项目摘要 我请求资金购买原子力显微镜(AFM)，以更换老化和故障的AFM 需要频繁的大修。原子力显微镜是父R35拨款中提议的工作的重要方法，它将 提供基于DNA的新工具，这些工具将克服与MP结构威慑相关的几个挑战 用单分子冷冻EM进行离子交换。 我们的DNA-脂质纳米盘的开发阶段需要原子力显微镜，因为原子力显微镜产生个体的图像 与其他成像方法相比，粒子具有更高的分辨率、对比度和信噪比 方法提供地形信息。在我们的实验室里可以立即使用这种仪器，避免了长时间的等待 因此缩短了我们基于DNA的工具的迭代改进周期。原子力显微镜样品制备是 与电子显微镜网格样品制备相比，速度快得多，需要的材料更少，同时避免染色、干燥 然后转移到吸尘器里。在AFM中，取而代之的是在本地缓冲区中对样本进行成像。这台新仪器支持 使用视频帧速率进行成像，这将使我们能够研究动态过程，包括纳米盘融合和 脂肪交换。此外，加速的成像大大减少了成像所需的时间 结构参数的生物物理量化所需的统计相关粒子数 以及DNA环的合成结果。最后，所要求的仪器将是世界上第一台现代原子力显微镜 因此，我期待着有趣的合作请求，这将利用我们在AFM方面的专业知识。

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.