Single-molecule fluorescence spectroscopy to study structure and conformational dynamics of model proteins and the transporter BetP from Corynebacterium glutamicum

虽然许多蛋白质都知道静态结构，但它们的功能往往受其动态特性的支配。在这个方案中，我们希望建立基于单分子、多参数检测和荧光相关光谱(FCS)的新的荧光光谱方法。我们希望发展新的稳健的滤波FCS(FFC)概念，以利用荧光各向异性和/或荧光共振能量转移(FRET)在宽时间范围(50 ns-10 ms)内研究蛋白质的构象动力学。我们将使用和验证这些新工具来监测选定的模型蛋白(例如DNA聚合酶I的T4溶菌酶和Klenow片段)中的构象交换。因此，我们的结果将与其他技术的结果进行比较。这项初步工作将使我们能够分析谷氨酸棒杆菌同源三聚体转运蛋白BetP的复杂结构和构象动力学。BetP是转运体功能分析的理想模型系统，因为对转运体的功能和结构有广泛的知识背景。然而，BetP的传感和转运机制仍不清楚。在研究脂质体中重组的单个BetP分子时，将解决以下问题：(I)分析其C-末端结构域在传感和催化过程中的构象变化；(Ii)催化循环中功能中间体的鉴定和结构表征；以及(Iii)通过监测固定化蛋白脂质体的FRET轨迹来建立催化循环的全貌。

Although static structures are known for many proteins, their functions are often governed by their dynamic character. In this proposal we want establish new fluorescence spectroscopic methods based on single molecules, multiparameter detection and fluorescence correlation spectroscopy (FCS). We want to develop new robust concepts of filtered FCS (fFCS) to study in a wide time range (50 ns -10 ms) the conformation dynamics of proteins using fluorescence anisotropy and/or fluorescence resonance energy transfer (FRET). We will use and verify these new tools to monitor conformational exchange in selected model proteins (e.g. T4 Lysozyme and Klenow Fragment of DNA Polymerase I). Thereby our results will be compared with those of other techniques. This preliminary work will enable us to analyze the complex structure and conformational dynamics of the homotrimer transporter BetP from Corynebacterium glutamicum. BetP is an ideal model system for the functional analysis of a transporter, because there is a broad background of knowledge on its function and structure available. Nevertheless the mechanisms for sensing and transport by BetP are still not clear. Studying single BetP molecules reconstituted in liposomes, the following questions will be addressed: (I) Analysis of the conformational changes of its C-terminal domain during sensing and catalysis; (II) Identification and structural characterization of functional intermediates in the catalytic cycle; and (III) Establishment of a full view of the catalytic cycle by monitoring FRET trajectories of immobilized proteoliposomes.