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

虽然许多蛋白质的静态结构是已知的，但它们的功能通常由它们的动态特性决定。本论文的目标是建立基于单分子、多参数检测和荧光相关光谱的新的荧光光谱分析方法。我们希望开发新的强大的概念过滤FCS（fFCS）的研究在一个宽的时间范围内（50 ns-10 ms）的构象动力学的蛋白质使用荧光各向异性和/或荧光共振能量转移（FRET）。我们将使用和验证这些新工具来监测选定的模型蛋白质（例如T4溶菌酶和DNA聚合酶I的Klenow片段）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。这一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是一个理想的模型系统的功能分析的转运，因为有一个广泛的背景知识，其功能和结构。然而，BetP的感知和运输机制仍然不清楚。研究单个BetP分子在脂质体中重组，将解决以下问题：（I）在传感和催化过程中其C-末端结构域的构象变化的分析;（II）在催化循环中的功能中间体的鉴定和结构表征;和（III）通过监测固定化的蛋白脂质体的FRET轨迹建立一个完整的催化循环视图。

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.