Detecting Individual Protein Conformation in Live Cells

描述（由申请人提供）：该提案旨在开发使用来自活细胞内蛋白质的单分子荧光（或福斯特）共振能量转移（sm FRET）的新能力，以观察真实的时间构象动力学，同时观察单个蛋白质在细胞网络内循环时的空间定位。跟踪单个分子的实验可以揭示由于分子和时间的固有平均以及缺乏同步而在批量测量中不可能观察到的特性。单粒子测量单个分子的动态构象可以确定多个反应途径和瞬态中间态以及分子性质的精确分布。在这个建议中的一般方法是使用高分辨率的结构信息已经为研究中的蛋白质设计位点特异性标记突变体。这些突变体将在无细胞的情况下产生、纯化和标记，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用于跟踪单个蛋白质分子，以及确定分子在活细胞内循环时的真实的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都无法观察到的细胞网络的细节。该方法将应用于2种不同的生理网络中，以确定：1）参与胞内囊泡转运和膜融合的SNARE蛋白中折叠/解折叠转变的空间分布和详细序列，以及2）第二信使信号传导中蛋白激酶A的激活。对细胞调节和信号网络的理解的进展将对健康相关研究的所有领域产生广泛的影响：对疾病状态，稳态和发展的理解。最终，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.