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

通过 NMR 光谱和 PET 荧光猝灭研究 ABC 输出体的构象动力学

• 批准号: 421388231
• 负责人: Professorin Dr. Ute Hellmich
• 金额: --
• 依托单位: Institut für Organische Chemie und Makromolekulare Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/421388231?language=en
• 关键词: Conformational; dynamics; ABC; exporter; investigated

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.

ATP 结合盒 (ABC) 转运蛋白是跨膜蛋白质组件，可实现各种分子化合物（例如有机小分子、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能导致耐药性，而转运蛋白功能障碍可能导致严重的疾病。然而，ABC 转运蛋白功能机制的分子细节仅被部分了解。构象变化的本质和顺序目前尚不清楚，构象变化是由所谓的核苷酸结合域 (NBD) 处的 ATP 结合和水解引发的，并导致底物通过跨膜域 (TMD) 运输。变构通讯的途径仍有待探索。在这里，我们将 NMR 光谱与光诱导电子转移 (PET) 荧光猝灭结合起来，这是蛋白质动力学中两种互补的高分辨率工具，以获得对乳酸乳球菌 LmrA ABC 输出蛋白功能机制的新见解。 NMR 光谱可产生有关蛋白质构象和动力学的原子信息，而 PET 荧光光谱可探索局部或远程结构元素的相关运动，并提供构象变化的时间常数。我们开始对高度保守的 NBD 进行单独研究，通过 NMR 绘制出核苷酸结合的动态后果，包括弛豫色散和 1H/2H 交换测量，以及具有纳秒时间分辨率的不同结构元件的探针重新配置时间常数将 PET 与荧光相关光谱 (PET-FCS) 结合使用。接下来，我们将在全长 ABC 输出器的背景下，使用 19F NMR 实验和 NBD 的 PET-FCS 确定与跨膜结构域 (TMD) 相互作用对 NBD 动力学的影响。最后，我们将利用定制的 PET 荧光报告基因设计来探测由核苷酸结合触发的 NBD 的二聚化以及 TMD 螺旋的整体倾斜运动，从而使配体易位。我们预计，我们的项目结果首次将 NMR 和 PET 荧光光谱作为两种互补的高分辨率解决方案技术相结合，为 ABC 转运蛋白的功能动力学提供新的见解，从而增强我们对结构和动力学的理解。这一类重要的膜蛋白中底物运输的基础。