Dynamics and Function of Spin Labelled ABC Transport System Studied by Multi-Frequency EPR

目前的ABC转运蛋白转运模型基本上是建立在对大肠杆菌/沙门氏菌麦芽糖转运蛋白MalFGK2的生化、生物物理和结构研究的基础上。由于这一系统的一些独特特点，这种运输模式是否适用于所有ABC运输商是值得怀疑的。这里，鼠伤寒沙门氏菌的组氨酸ABC转运蛋白HisJ/LAO-HisQMP2和ECF(能量偶联因子)转运蛋白家族的生物素转运蛋白BioMNY被认为是ABC转运蛋白的互补模型系统。根据我们最近的研究结果，我们现在将集中在底物结合蛋白HisJ和HisQMP2之间的相互作用以及底物和核苷酸诱导的ECF转运蛋白的构象变化。多频连续波谱和脉冲EPR谱将跟踪这些转运蛋白的定点自旋标记变体在转运过程中与功能相关的构象变化，目的是在分子水平上揭示转运机制(S)。此外，我们打算在EPR波谱领域开发和应用新的方法学方法，包括基于分子动力学模拟的EPR波谱计算。

The current transport model of ABC transporters is essentially based on the biochemical, biophysical and structural investigations of the maltose transporter MalFGK2 of E. coli/Salmonella. Due to some unique features of this system it is questionable whether this transport model holds for all ABC transporters. Here, the histidine ABC transporter from Salmonella typhimurium, HisJ/LAO-HisQMP2, and of the ECF (“energy-coupling factor”) transporter family, the biotin transporter BioMNY are regarded as complementary model systems for ABC transporters. Based on our recent results we will focus now on the interaction between the substrate binding protein HisJ and HisQMP2 and on substrate and nucleotide induced conformational changes of ECF transporters. The functionally related conformational changes of site-directed spin labeled variants of these transporters will be followed during the transport cycle by multi-frequency cw and pulse EPR spectroscopy with the aim to unravel the transport mechanism(s) on molecular level. In addition we intend to develop and apply new methodological approaches in the field of EPR spectroscopy including MD simulation based EPR spectra calculations.