Conformational dynamics of an ABC exporter investigated by NMR spectroscopy and PET fluorescence quenching

ATP结合盒（ABC）转运蛋白是跨膜蛋白组装体，其实现各种分子化合物（诸如小有机分子、糖、肽或离子）跨活细胞膜的主动转运。ABC转运蛋白具有医学相关性，因为它们的ATP酶驱动的转运机制可导致耐药性，并且转运蛋白功能障碍可导致严重疾病。然而，ABC转运蛋白的功能机制的分子细节仅被部分理解。由ATP结合和水解在所谓的核苷酸结合结构域（NBD）处触发并导致底物通过跨膜结构域（TMD）转运的构象变化的性质和序列目前尚不清楚。变构通讯的途径仍有待探索。在这里，我们结合联合收割机NMR光谱与荧光猝灭的光诱导电子转移（PET），蛋白质动力学的两个互补的高分辨率工具，获得新的见解的功能机制的LmrA ABC出口从L。乳酸。虽然NMR光谱产生蛋白质构象和动力学的原子信息，PET荧光光谱探索跨本地或远程结构元素的相关运动，并提供构象变化的时间常数。我们开始我们的调查高度保守的NBD在隔离，在那里我们绘制出的动态后果的核苷酸结合NMR，包括弛豫分散和1H/2 H交换测量，和探针重构时间常数的不同的结构元素与纳秒时间分辨率使用PET结合荧光相关光谱（PET-FCS）。接下来，我们将使用19 F NMR实验和PET-FCS的NBD在全长ABC出口商的上下文中确定与跨膜结构域（TMD）的相互作用对NBD动力学的影响。最后，我们将探测由核苷酸结合引发的NBD二聚化和使用定制的PET荧光报告设计易位配体的TMD螺旋的全局倾斜运动。我们预计，我们的项目的结果，其中NMR和PET荧光光谱作为两个互补的高分辨率的解决方案技术相结合的第一次，提供了新的见解ABC转运蛋白的功能动力学，从而提高我们的理解在这类重要的膜蛋白底物运输的结构和动力学基础。

ATP binding cassette (ABC) transporters are trans-membrane protein assemblies that accomplish the active transport of various molecular compounds, such as small organic molecules, sugars, peptides or ions, across the membrane of living cells. ABC transporters are of medical relevance because their ATPase-driven transport mechanism can lead to drug resistance and transporter dysfunctions can cause severe diseases. The molecular details of the functional mechanism of ABC transporters, however, are only partially understood. The very nature and sequence of conformational changes, which are triggered by ATP binding and hydrolysis at the so-called nucleotide binding domains (NBDs) and lead to transport of a substrate through the transmembrane domains (TMDs), are currently unclear. Pathways of allosteric communication remain to be explored. Here, we combine NMR spectroscopy with fluorescence quenching by photoinduced electron transfer (PET), two complementary high-resolution tools in protein dynamics, to gain new insights into the functional mechanism of the LmrA ABC exporter from L. lactis. While NMR spectroscopy yields atomistic information on protein conformation and dynamics, PET fluorescence spectroscopy explores correlated motions across local or remote structural elements and delivers time constants of conformational change. We start our investigation on the highly-conserved NBD in isolation, where we map out the dynamic consequences of nucleotide binding by NMR, including relaxation-dispersion and 1H/2H exchange measurements, and probe reconfiguration time constants of distinct structural elements with nanosecond time resolution using PET in combination with fluorescence correlation spectroscopy (PET-FCS). Next, we will determine the consequences of interactions with the trans-membrane domain (TMD) on the dynamics of the NBD using 19F NMR experiments and PET-FCS of the NBD in context of the full-length ABC exporter. Finally, we will probe dimerization of NBDs triggered by nucleotide binding and the global tilting motions of TMD helices that translocate a ligand using a tailored PET fluorescence reporter design. We anticipate that the results of our project, where NMR and PET fluorescence spectroscopy as two complementary high-resolution solution techniques are combined for the first time, provide new insights into the functional dynamics of ABC transporters and thereby enhance our understanding of the structural and dynamical basis of substrate transport in this important class of membrane proteins.