Detecting Individual Protein Conformation in Live Cells

描述(申请人提供)：这项建议旨在开发一种新的能力，利用活细胞内蛋白质的单分子荧光(或Forster)共振能量转移(Sm Fret)来观察单个蛋白质在细胞网络中循环时的实时构象动态和空间定位。跟踪单个分子的实验可以发现由于分子和时间的固有平均以及缺乏同步性而不可能在整体测量中观察到的性质。单粒子测量单个分子的动态构象可以确定多条反应路径和瞬时中间态，以及分子性质的准确分布。这项建议中的一般方法是利用研究中蛋白质已有的高分辨率结构信息来设计特定位置的标记突变体。这些突变体将被生产、纯化并标记为无细胞，然后显微注射到培养的真核细胞中。荧光显微镜和光谱学将被用来跟踪单个蛋白质分子，以及当它们在活细胞内循环时实时确定该分子的FRET程度。这种未经测试的方法如果成功，将对许多生物学问题具有广泛的适用性，并有可能揭示任何其他技术无法观察到的细胞网络的细节。这一方法将在两个不同的生理网络中应用，以确定1)参与细胞内囊泡运输和膜融合的SNARE蛋白折叠/去折叠转换的空间分布和详细序列，以及2)第二信使信号中蛋白激酶A的激活。在了解细胞调节和信号网络方面的进展将对健康相关研究的所有领域产生广泛影响：了解疾病状态、体内平衡和发育。归根结底，通过这些方法获得的知识将改善人类健康。

DESCRIPTION (provided by applicant): This proposal aims to develop the new capability of using single molecule fluorescence (or Forster) resonance energy transfer (sm FRET) from proteins within living cells to observe real time conformational dynamics in parallel with spatial localization of individual proteins as they circulate within cellular networks. Experiments that follow single molecules can uncover properties that are impossible to observe in bulk measurements due to the inherent averaging over molecules and over time, and lack of synchronization. Single particle measurements of dynamic conformations of individual molecules can determine multiple reaction pathways and transient intermediate states as well as exact distributions of molecular properties. The general approach in this proposal is to use high resolution structural information already available for the proteins under study to design site-specific labeling mutants. These mutants will be produced, purified and labeled free of cells and then microinjected into cultured eukaryotic cells. Fluorescence microscopy and spectroscopy will be used to track individual protein molecules as well as determine the degree of FRET from that molecule in real time as they circulate within living cells. This untested approach, if successful, will have broad applicability to many biological questions and has the potential to reveal details of cellular networks that are not observable by any other technique. This approach will be applied within 2 different physiological networks to determine, 1) the spatial distribution and detailed sequence of folding/unfolding transitions in SNARE proteins involved in intracellular vesicle transport and membrane fusion, and 2) the activation of Protein Kinase A in second messenger signaling. Advances in understanding of cellular regulatory and signaling networks will have broad impact in all areas of health related research: understanding of the disease state, homeostasis and development. Ultimately knowledge derived with these methods will improve human health.