Bridge 3: The Transport Cycle in Neurotransmitter Uptake Systems

次级活性转运体使用来自电化学离子梯度和/或底物梯度的能量来 在所有生物体的生物膜上调节浓缩底物的转移。随着技术的进步 膜蛋白结构生物学研究表明，存在大量的次级活性共转运蛋白 然而，属于没有可辨别的序列同一性的远亲家庭的交换者，分享 将它们归类为单个结构族的常见结构特征，称为Leut-Fold。这个褶皱 其特征是10个跨膜螺旋(TM)被组织成两个倒置结构重复序列 含5个TM。在桥3中，我们试图了解功能组件中的共性和差异 Leut-折叠蛋白质的机制，重点是蛋白质结构动力学的一般规则， 基础功能，在它们的驱动机制和构象变化的不同背景下 与底物移位有关。为了实现这些目标，我们建立在我们已有的协同方法之上 与Leut的研究一起建立，使用迭代计算、泛函和光谱方法。 这座桥的工作假设是，机制差异的发现和解释 Leut和其他Leut折叠运输器之间的关系取决于揭示由本地实现的动态属性 结构性差异。我们将使用新一代定量计算方法，与 结合和助熔剂研究，并用EPR和单分子荧光研究来测量 不同构象状态的探针对以及相关运动的动力学。这个 工作将利用团队成员之间建立的特定协作，并与 计算建模、光谱和仪器，以及蛋白质表达核心。 ApcT是APC家族的成员，其中还包括产品/前体交易所，该公司共享Leut-Fold，但 已被报道是一种H+依赖的氨基酸转运体。有趣的是，ApcT具有侧链ε-氨基 占据了Leut的Na2位的Lys158群，已经被认为质子化和 这种赖氨酸的去质子化，就像亮氨酸中Na2的结合和解离一样，驱动着运输。与之相对的是 ApcT和Leut之间的深刻机制差异，果蝇多巴胺转运体(DDAT)是 在整体结构和功能上与Leut密切相关，但由于存在大的氨基酸和 已被证明在真核生物中关键地调节转运蛋白功能的羧基末端 传送者。为了了解这些比较的Leut在作用机制上的异同- 折叠转运蛋白我们提出了以下具体目标：1)确定底物、H+和Na+如何 ApcT转运循环中的配位动力学和构象变化 莱特。2)整合CW和鹿的测量，以探索氨基末端在 调控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.