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组件，可在整个活细胞的膜上完成各种分子化合物的主动转运，例如小有机分子，糖，肽或离子。 ABC转运蛋白具有医学相关性，因为它们的ATPase驱动的运输机制可以导致耐药性，而转运蛋白功能障碍可能导致严重的疾病。但是，仅部分了解了ABC转运蛋白功能机理的分子细节。构象变化的本质和序列是由所谓的核苷酸结合结构域（NBD）触发的，这些变化是由ATP结合和水解触发的，并导致底物通过跨膜结构域（TMDS）传输。变构沟通的途径仍有待探索。在这里，我们将NMR光谱与光诱导的电子转移（PET）（PET）（蛋白质动力学中的两种互补的高分辨率工具）结合起来，以获得对L. lactis L. L. l. abc Exporter的功能机理的新见解。虽然NMR光谱验证了有关蛋白质构象和动力学的原子信息，但宠物荧光光谱探索了局部或远程结构元素的相关运动，并提供了构象变化的时间常数。 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).接下来，我们将使用19F NMR实验和NBD的PET-FC在全长ABC出口商的情况下确定与反膜域（TMD）相互作用对NBD的动力学的相互作用。最后，我们将探测由核苷酸结合触发的NBD和TMD螺旋的全局倾斜运动触发的，这些螺旋可以使用量身定制的PET荧光报道器设计转移配体。我们预计，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的结果，首次合并了NMR和PET荧光光谱法，为ABC转运蛋白的功能动力学提供了新的见解，从而增强了我们对在膜蛋白的这一重要类别中基质运输的结构和动态基础的理解。