Bridge 3: The Transport Cycle in Neurotransmitter Uptake Systems

次级主动转运蛋白使用来自电化学离子梯度和/或底物梯度的能量， 介导所有生物体的生物膜的集中底物转运。的进步 膜蛋白结构生物学，它已成为明确的，大量的二级活性共转运蛋白 和交换器，属于遥远的家庭，没有可辨别的序列同一性，然而，共享 共同的结构特征，将它们分为一个单一的结构家族，称为LeuT折叠。这圈 其特征在于10个跨膜螺旋（TM），每个TM组织成两个反向结构重复， 包含5个TM。在Bridge 3中，我们试图理解功能性的共性和分歧， LeuT折叠蛋白的机制，重点是蛋白质结构动力学的一般规则， 在驱动机制和构象变化差异的背景下， 与底物转运有关。为了实现这些目标，我们建立在协同的方法，我们有 建立与LeuT的研究，采用迭代计算，功能和光谱方法。 这座桥的工作假设是，发现和解释机械差异 LeuT和其他LeuT折叠转运蛋白之间的联系依赖于揭示局部 结构性差异。我们将使用新一代的定量计算方法，与 结合和通量研究，并与EPR和单分子荧光研究，以测量之间的距离 不同构象状态的探针对以及相关运动的动力学。的 工作将利用团队成员之间建立的具体合作，并与 计算建模，光谱和仪器，蛋白质表达核心。 ApcT是APC家族的一员，也包括产物/前体交换剂，其共享LeuT折叠，但 据报道是H+依赖性氨基酸转运蛋白。有趣的是，ApcT具有侧链ε-氨基 一组Lys 158占据LeuT中的Na 2位点，并且有人认为质子化和 该Lys的去质子化，如LeuT中Na 2的结合和解结合，驱动运输。相对于 ApcT和LeuT之间存在深刻的机制差异，果蝇多巴胺转运蛋白（dDAT）是 在整体结构和功能上与LeuT密切相关，但因存在大的氨基和 羧基末端，其已被证明在真核生物中关键地调节转运蛋白功能。 运输机为了了解这些比较LeuT功能机制的异同， 我们提出以下具体目的：1）确定底物，H+和Na+ 在运输循环中的协调动力学和构象变化的ApcT相比， LeuT。2)整合CW和DEER测量，探索氨基末端在 调节dDAT的构象动力学。

Secondary active transporters use the energy from electrochemical ion gradients and/or substrate gradients to mediate concentrative substrate translocation across biological membranes of all organisms. With advances in membrane protein structural biology, it has become clear that a large number of secondary active symporters and exchangers, which belong to distant families without discernable sequence identity, nonetheless, share common structural features that classify them into a single structural family, known as the LeuT-fold. This fold is characterized by 10 transmembrane helices (TMs) organized into two inverted structural repeats each containing 5 TMs. In Bridge 3 we seek to understand commonalities as well as divergence in the functional mechanisms of the LeuT-fold proteins, with a focus on the general rules of protein structural dynamics that underlie function, in the context of differences in their driving mechanism and the conformational changes associated with substrate translocation. To achieve these goals we build on the synergistic approach we have established with the study of LeuT, employing iterative computational, functional and spectroscopic methods. The working hypothesis of this Bridge is that the discovery and interpretation of mechanistic differences between LeuT and other LeuT-fold transporters depends on revealing dynamic properties enabled by local structural differences. We will use a new generation of quantitative computational approaches, in parallel with binding and flux studies, and with EPR and single-molecule fluorescence studies to measure distance between pairs of probes in different conformational states as well as the dynamics of the associated movements. The work will take advantage of specific established collaborations among the team members, and with the Computational Modeling, Spectroscopy and Instrumentation, and Protein Expression Cores. ApcT, a member of the APC family that also includes product/precursor exchangers, shares the LeuT-fold but has been reported to be a H+-dependent amino acid transporter. Interestingly, ApcT has the side chain ε-amino group of Lys158 occupying what is the Na2 site in LeuT, and it has been suggested that protonation and deprotonation of this Lys, like binding and unbinding of Na2 in LeuT, drives transport. In contrast to the profound mechanistic differences between ApcT and LeuT, the Drosophila dopamine transporter (dDAT) is closely related to LeuT in overall structure and function but differentiated by the presence of large amino and carboxy termini, which have been shown to critically modulate transporter function in the eukaryotic transporters. To understand similarities and differences in functional mechanisms for these compared LeuT- fold transporters we propose the following Specific Aims: 1) To determine how substrates, H+ and Na+ coordinate dynamics and conformational changes in the transport cycle of ApcT as compared with LeuT. 2) To integrate CW and DEER measurements in exploring the role of the amino terminus in modulating the conformational dynamics of dDAT.