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。本项目的重点是大肠杆菌的乳糖渗透酶转运蛋白(Lacy)。Lacy通过细胞膜运输特定的糖分子和质子，是典型的膜蛋白，它催化糖等营养分子的运输，而不是浓度梯度。这个项目的目标是准确地了解这种耦合机制是如何工作的。目前的生化/生物物理研究为Lacy的糖和质子结合部位交替进入膜两侧的机制提供了支持证据，这是蛋白质整体结构变化的结果。虽然交替通道现在被普遍认为是膜运输的机制，但糖和质子运输之间的耦合化学仍未解决。目前的研究项目将利用一些骆驼纳米体，这是一种特殊形式的抗体，以稳定不同中间状态下的Lacy，这将首次为这类运输蛋白的机制提供深入的了解。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，并为系统地处理难以研究的膜蛋白的结构决定提供一个模型。该项目将为研究生和本科生提供培训和教育。本研究的目的是在原子水平上了解乳糖/质子转运的机制，通过乳糖渗透酶(Lacy)，这是一个范例的主要促进者超家族(MFS)，最大的膜运输蛋白。MFS的成员存在于所有活细胞的细胞膜中。然而，尽管MFS成员的X射线结构越来越多，而且有证据表明乳糖/质子结合是由化学渗透作用热力学驱动的，但这种化学渗透过程的机制并不完全清楚。因此，已经证明糖与高动态和质子化的Lacy结合会触发全球构象变化，其中糖和质子结合位置交替进入膜的两侧，但很明显，糖的结合和解离通过诱导Fit机制驱动这种构象变化，而质子电化学梯度加快了去质子化的速度。因此，花边的行为很像一种酶，除了过渡态(S)涉及蛋白质而不是底物。Lacy向内构象和几乎被遮挡的向外构象的X射线结构为研究交替存取机制提供了结构基础。将通过应用稳态前动力学以及PI实验室首创的多种生化和光谱方法，并通过使用运输周期中几个步骤的实时动力学数据来研究交替访问机制。本研究的重点是利用驼绒纳米体来稳定不同中间态的花边，并用X射线衍射法对其进行研究。这些研究将使我们对共生机制有更深入的了解。

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.