EAGER: Mechanism of Energy Coupling with a Membrane Symport Protein

糖、氨基酸和其他营养物质跨细胞膜运输的详细机制是一个尚未解决的生物学问题。该项目的重点是乳糖通透酶转运蛋白（LacY）的细菌大肠杆菌。LacY转运特定的糖分子和质子穿过细胞膜，是典型的催化营养分子如糖逆浓度梯度转运的膜蛋白。这个项目的目标是准确地理解这种耦合机制是如何工作的。目前的生物化学/生物物理学研究提供了支持证据的机制，其中糖和质子结合位点的LacY获得交替进入膜的两侧作为全球结构变化的结果在蛋白质。尽管现在人们普遍认为交替进入是膜运输的机制，但糖和质子运输之间偶联的化学反应仍然没有得到解决。目前的研究项目将利用一些骆驼科纳米抗体，这是一种特殊形式的抗体，以稳定LacY在不同的中间状态，这将提供深入了解的机制首次为这类转运蛋白。了解这一机制的分子细节将解决生命系统功能的一个重要问题，并提供一个模型，系统地处理膜蛋白的结构决定，是很难研究。 该项目将向研究生和本科生提供培训和教育。本研究的目的是开发一个原子级的理解乳糖/质子共转运的机制，乳糖通透酶的大肠杆菌（LacY），一个范例的主要促进剂超家族（MFS），最大的家庭膜转运蛋白。MFS的成员存在于所有活细胞的膜中。然而，尽管越来越多的X射线结构的MFS成员，以及证明乳糖/质子共转运是由化学渗透驱动的，这种化学渗透过程的机制还没有完全理解。因此，它已被证明，糖结合到高度动态和质子化的LacY触发一个全球性的构象变化，其中糖和质子结合位点获得交替进入膜的两侧，但很明显，糖的结合和解离驱动这种构象变化通过诱导配合机制，而质子电化学梯度加速去质子化的速率。因此，LacY的行为很像一种酶，除了过渡态涉及蛋白质而不是底物。X射线结构的LacY向内和几乎闭塞面向外的构象提供了研究交替访问机制的结构基础。交替访问机制将通过应用前稳态动力学，以及在PI的实验室开创的多种生物化学和光谱方法，并通过使用动力学数据在运输周期的几个步骤中获得的真实的时间进行研究。这项研究的重点是使用骆驼纳米抗体来稳定LacY在不同的中间状态，通过X射线衍射进行研究。这些研究将提供一个深入的了解共转运机制。

The detailed mechanism of the transport of sugars, amino acids and other nutrients across cell membranes is an unsolved biological problem. This project focuses upon the lactose permease transporter (LacY) of the bacterium Escherichia coli. LacY transports a specific sugar molecule and a proton across the cell membrane and is typical of membrane proteins that catalyze the transport of nutrient molecules such as sugars against a concentration gradient. The goal of this project is to understand precisely how this coupled mechanism works. Current biochemical/biophysical studies provide supporting evidence for a mechanism in which sugar- and proton-binding sites of LacY gain alternating access to either side of the membrane as the result of global structural changes in the protein. Although alternating access is now generally accepted as the mechanism for membrane transport, the chemistry of coupling between sugar and proton transport remains unresolved. The current research project will utilize a number of Camelid nanobodies, which are a special form of antibody, to stabilize LacY in different intermediate states that will provide an in-depth understanding of the mechanism for the first time for this class of transport proteins. Understanding the molecular details of this mechanism will address an important issue for the function of living systems and provide a model for systematically dealing with structural determinations of membrane proteins that are difficult to study. The project will provide training and education to students at the graduate and undergraduate level. The aim of this research is to develop an atomic-level understanding of the mechanism of lactose/proton symport by the lactose permease of Escherichia coli (LacY), a paradigm for the Major Facilitator Superfamily (MFS), the largest family of membrane transport proteins. Members of the MFS are found in the membranes of all living cells. However, despite an increasing number of X-ray structures of MFS members, as well as the demonstration that lactose/proton symport is driven thermodynamically by chemiosmosis, the mechanism of this chemiosmotic process is not completely understood. Thus, it has been demonstrated that sugar binding to highly dynamic and protonated LacY triggers a global conformational change in which sugar- and proton-binding sites gain alternating access to either side of the membrane, but it is apparent that sugar binding and dissociation drive this conformational change through an induced-fit mechanism, while the proton electrochemical gradient accelerates the rate of deprotonation. Therefore, LacY behaves much like an enzyme except that the transition state(s) involves the protein rather than the substrate. X-ray structures of LacY inward- and almost occluded outward-facing conformations provide the structural basis for studying the alternating access mechanism. The alternating access mechanism will be studied by applying pre-steady state kinetics, as well as multiple biochemical and spectroscopic approaches pioneered in the PI's laboratory and by using kinetic data obtained in real time for several steps in the transport cycle. This research focuses on the use of Camelid nanobodies to stabilize LacY in different intermediate states to be studied by X-ray diffraction. These studies will provide an in-depth understanding of the symport mechanism.