Development of Rotational Fluctuation Spectroscopy for Biological Applications

描述(由申请人提供)：本提案是根据RFA-RR-05-001提交的，描述了一种创新的方法学--旋转荧光相关光谱学的发展，用于在广泛的生物医学应用中测量分子相互作用。细胞环境中分子间的相互作用是当代细胞生物学的重要研究领域。自1972年引入以来，荧光相关光谱(FCS)已被证明是测量生物分子平移扩散以及相互作用的有力工具。然而，蛋白质的旋转运动，由于受到扩散物种的大小和环境的调节，是这种相互作用的一个比横向扩散更敏感的探针。与FCS在测量平移扩散方面的广泛应用相比，FCS在分子转动研究中的应用还不是很广泛。我们的实验室专注于分子旋度的测量，使用和开发用于此目的的方法，并在这一领域发表的所有工作中贡献了相当大的一部分。在这个项目中，我们将探索实现旋转荧光相关光谱所涉及的理论和实验问题，并将开发应用于溶液中和完整细胞中的分子的实用策略。我们的具体目标是开发一个实用的基于双光子激发的旋转荧光相关光谱系统(目标1)，开发从旋转FCS数据中分离粒子旋转和平移动力学的数据分析方法(目标2)，并评估旋转FCS方法在测量选定测试系统的旋转动力学方面的性能(目标3)。该项目的创新方面包括在相关数据中分离旋转和平移动力学的新战略，将旋转FCS测量扩展到观察溶解蛋白质之间相互作用的纳秒时间尺度，以及实现旋转FCS数据的在线分析。此外，将构建的多目标区域探测器将首次允许从显微镜观察中评估真实的发光发射各向异性，而不需要引入关于被观测分子运动约束的先验假设。

DESCRIPTION (provided by applicant): This proposal, submitted in response to RFA-RR-05-001, describes development of an innovative methodology, rotational fluorescence correlation spectroscopy, for measurement of molecular interactions in a wide range of biomedical applications. Interactions between molecules in cellular environments are key areas of interest in contemporary cell biology. Since its introduction in 1972, fluorescence correlation spectroscopy (FCS) has proved a powerful tool for measuring translational diffusion, and thus interactions, of biological molecules. However, protein rotational motion, as regulated by the size and environment of the diffusing species, is a much more sensitive probe of such interactions than is lateral diffusion. In contrast with its wide-spread application in measuring translational diffusion, FCS has not been applied in a significant way to examination of molecular rotation. Our laboratory focuses on measurement of molecular rotation, using and developing methods for this purpose, and has contributed a substantial fraction of all work published in this area. In this project, we will explore the theoretical and experimental issues involved in implementing rotational fluorescence correlation spectroscopy and will develop of practical strategies for application to molecules in solution and in intact cells. Our specific aims are to develop a practical microscope-based system for rotational fluorescence correlation spectroscopy based on two-photon excitation (Aim 1), to develop data analysis methods for separating rotational and translational dynamics of particles from rotational FCS data (Aim 2) and to evaluate the performance of rotational FCS methods in measuring rotational dynamics of selected test systems (Aim 3). Innovative aspects of the project include new strategies for separating rotational and translational dynamics in correlation data, for extending rotational FCS measurement into the nanosecond timescale where interactions between dissolved proteins are observed and for achieving on-line analysis of rotational FCS data. Moreover, the multi-objective region detector to be constructed will, for the first time, permit true luminescence emission anisotropy to be evaluated from microscope observations without introducing a priori assumptions about motional constraints of observed molecules.