AFM(T) + FLUORESCENCE MEASUREMENTS ON SINGLE MOLECULES

描述（由申请人提供）：我们的长期目标是增加选择健康和疾病过程的分子水平的理解。我们寻求实现这一目标的一个新的仪器，能够单分子荧光测量的角度（偏振），平移，或构象运动收集之前，期间，或之后AFM成像和操作的个别生物分子及其复合物。原子力显微镜是唯一的仪器，可以图像湿，天然样品与纳米分辨率，它也已成为一个关键技术的操纵型研究的单分子和复合物，包括研究酶的活性在压缩限制和蛋白质折叠展开张力下开始由Gaub和费尔南德斯。Discher实验室是第一个通过AFM（Temp）在蛋白质强制延伸期间证明单一蛋白质稳定性的强烈温度效应的实验室，最近表明蛋白质-蛋白质相互作用可以检测到与解折叠的耦合，但仍然存在大量的分子生物物理学问题。荧光显微镜在过去十年中也发展到单分子检测，应用于肌球蛋白马达的两项最新创新来自Goldman实验室和合作者，直接测量旋转运动以及将横向运动的空间分辨率提高到1 nm。库珀曼和戈德曼已经开始将这些方法应用于核糖体。Shuman实验室同样开发了相关的基于光学的技术，用于蛋白质-蛋白质相互作用的机械询问和分子马达中的能量转导。为了更深入地开展这项NIH资助的工作和其他项目，包括外部提交的项目（每年5周），我们建议广泛使用能够在AFM成像和操作之前，期间或之后进行单分子荧光的多用途仪器（T=20-60 ℃）。通过一些附件，荧光方法将包括双色偏振TIRF，单对FRET和nm级距离测量，而我们在同一区域使用AFM（T）对复合物和蛋白质进行成像或同时进行单分子机械测试。该工具的主要用户将调查至少7个NIH资助的与公共卫生相关的研究项目，包括：核糖体在压力和限制下的功能具有根本的重要性，肌球蛋白粗丝在与心肌病相关的力下，微小肌营养不良蛋白和血影蛋白延伸性与肌营养不良和贫血的基因治疗的生物物理基础相关，以及与免疫系统应答相关的整联蛋白相关蛋白的细胞膜粘附。

DESCRIPTION (provided by applicant): Our long-term objectives are to increase molecular-level understanding of select health and disease processes. We seek to achieve this with a new instrument capable of single-molecule fluorescence measurements of angular (polarization), translational, or conformational motions collected before, during, or after AFM-imaging and manipulation of individual biomolecules and their complexes. AFM is the only instrument that can image wet, native samples with nanometer resolution, and it has also emerged as a key technology for manipulation-type studies of single molecules and complexes, including studies of enzyme activity under compressive confinement and protein folding-unfolding under tension begun by Gaub and Fernandez. The Discher lab was the first to demonstrate strong temperature effects on stability of single proteins during forced extension of proteins by AFM(Temp) and more recently showed that protein-protein interactions can detectably couple to unfolding but a large number of molecular biophysics questions remain. Fluorescence microscopy has also progressed over the last decade to single molecule detection, and two of the latest innovations applied to myosin motors come from the Goldman lab and collaborators with direct measurement of rotational motions as well as improved spatial resolution of lateral motions to 1 nm. Cooperman & Goldman have begun to apply these methods to ribosomes. The Shuman lab has likewise developed related, optics-based technology for mechanical interrogation of protein-protein interactions and for energy transduction in molecular motors. To more deeply pursue this NIH-funded work and additional projects, including externally submitted projects (5 wks per yr), we propose to make extensive use of a multi-purpose instrument capable of performing single molecule fluorescence before, during, or after imaging and manipulation by AFM(T=20-60 ¿C). With a few accessories, the fluorescence methods will include two-color Polarization-TIRF, single-pair FRET, and nm-scale distance measurements while we use AFM(T) on the same region to either image or simultaneously perform single molecule mechanical tests on complexes and proteins. Key users of the instrument will investigate at least 7 NIH-funded research projects that are relevant to public health including: ribosome function under stress and confinement of fundamental importance, myosin thick filaments under force relevant to cardiomyopathies, mini-dystrophin and spectrin extensibility relevant to biophysical foundations for gene therapy of both muscular dystrophy and anemias, and cell membrane adhesion of integrin-associated proteins relevant to immune system responses.