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对选定的健康和疾病过程的分子水平的理解。我们试图通过一种新的仪器来实现这一目标，该仪器能够在afm成像和单个生物分子及其复合物的操作之前，期间或之后收集单分子荧光测量角（偏振），平移或构象运动。AFM是唯一能够以纳米分辨率对湿的原生样品进行成像的仪器，它也成为单分子和复合物的操作型研究的关键技术，包括Gaub和Fernandez开始的压缩约束下酶活性和张力下蛋白质折叠展开的研究。Discher实验室首先证明了在AFM（Temp）强制拉伸蛋白质时，温度对单个蛋白质稳定性的强烈影响，最近又表明蛋白质-蛋白质相互作用可以检测到展开，但仍有大量的分子生物物理学问题有待解决。荧光显微镜在过去十年中也发展到单分子检测，两项应用于肌球蛋白马达的最新创新来自高盛实验室和合作者，直接测量旋转运动以及将横向运动的空间分辨率提高到1nm。Cooperman & Goldman已经开始将这些方法应用于核糖体。舒曼实验室同样开发了相关的光学技术，用于蛋白质相互作用的机械检测和分子马达的能量转导。为了更深入地开展这项美国国立卫生研究院资助的工作和其他项目，包括外部提交的项目（每年5周），我们建议在AFM成像和操作之前，期间或之后广泛使用能够执行单分子荧光的多用途仪器（T=20-60¿C）。加上一些附件，荧光方法将包括双色偏振- tirf，单对FRET和纳米尺度距离测量，而我们在同一区域使用AFM(T)成像或同时对复合物和蛋白质进行单分子力学测试。该工具的主要用户将调查至少7个美国国立卫生研究院资助的与公共卫生有关的研究项目，包括：核糖体在压力和限制下的功能具有根本的重要性，肌球蛋白在压力下的粗丝与心肌病有关，mini-dystrophin和spectrin的延伸与肌肉萎缩症和贫血的基因治疗的生物物理基础有关，以及与免疫系统反应相关的整合素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.