EAGER: Molecular Mechanism of Permeases

糖、氨基酸和其他营养物质跨细胞膜的运输是一个尚未解决的生物学问题。这个过程是由蛋白质介导的。LacY就是其中一种蛋白质，它参与乳糖和质子的运输。LacY以一种典型的膜蛋白的方式运作，催化运输对抗浓度梯度（即主动运输）。在这个过程中，在质子动力存在的情况下，LacY利用质子的下坡传输来驱动糖的上坡浓度。这个项目的目的是精确地理解这种耦合机制是如何工作的。PI在原子水平上获得了LacY晶体结构，生化/光谱研究表明，由于LacY两侧空腔的相互打开和关闭，糖和质子结合位点可以交替进入膜的两侧。本提案将通过使用晶体学方法研究LacY在不同输运周期状态下的结构方面。该项目还将为本科生和研究助理提供培训，让他们直接参与研究的实验方面。本研究的目的是在原子水平上了解大肠杆菌乳糖渗透酶（LacY）的乳糖/H+同调机制，这是主要促进物超家族（MFS）的一个范例，MFS是最大的膜运输蛋白家族。MFS成员存在于所有活细胞的细胞膜中。然而，尽管越来越多的MFS成员的x射线结构，包括来自PI实验室的7个LacY，以及定量证明乳糖/H+同质是由化学渗透驱动的热力学，但这种化学渗透过程的机制尚不完全清楚。因此，研究表明，半乳糖苷与高动态质子化LacY结合引发了一种全局构象变化，其中糖和质子结合位点交替进入膜的两侧，而质子电化学梯度加速了去质子化的速度，但对交替进入没有影响。因此，LacY的行为类似于酶，只是过渡状态涉及蛋白质而不是底物。LacY向内和几乎封闭的向外构象的x射线结构为研究交替存取机制提供了结构基础。PI计划通过应用预稳态动力学，以及多种生化和光谱方法，并通过在运输周期的几个步骤中实时获得的动力学数据来研究交替访问机制。PI计划通过确定交换和逆流是否受到该梯度与其他MFS同调子的影响来确定关于电化学梯度效应的发现是否可以推广。本研究还重点研究了利用31个骆驼类纳米体稳定不同中间态的LacY，并用x射线衍射研究。该项目得到了生物科学理事会分子和细胞生物科学部分子生物物理集群的支持。

Transport of sugars, amino acids and other nutrients across cell membranes is an unsolved biological problem. This process is mediated by proteins. LacY is one of such proteins and it is involved in the transport of lactose and protons. LacY operates in a manner typical of membrane proteins that catalyze transport against a concentration gradient (i.e. active transport). In this process, in the presence of a proton motive force, downhill transport of the protons is used by LacY to drive uphill concentration of sugar against a gradient. The aim of this project is to understand precisely how this coupled mechanism works. The PI has obtained LacY crystal structures at the atomic level, and biochemical/spectroscopic studies show that sugar- and proton-binding sites gain alternating access to either side of the membrane as the result of reciprocal opening and closing of cavities on either side of LacY. This proposal will investigate structural aspects of LacY at different states of the transport cycle by using crystallographic approaches. This project will also provide training to undergraduate students and research associates by directly involving them in experimental aspects of the research. The aim of this research is to develop an atomic-level understanding of the mechanism of lactose/H+ 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, including 7 of LacY from the PI's laboratory, as well as the quantitative demonstration that lactose/H+ symport is driven thermodynamically by chemiosmosis, the mechanism of this chemiosmotic process is not completely understood. Thus, it has been demonstrated that galactoside binding to highly dynamic protonated LacY triggers a global conformational change in which sugar- and proton-binding sites gain alternating access to either side of the membrane, while the proton electrochemical gradient accelerates the rate of deprotonation, but has no effect on alternating access. Therefore, LacY behaves 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 PI plans to study the alternating access mechanism by applying pre-steady state kinetics, as well as multiple biochemical and spectroscopic approaches, and by using kinetic data obtained in real time for several steps in the transport cycle. The PI plans to determine whether the findings regarding the effects of electrochemical gradient are generalized by determining whether exchange and counterflow are affected by this gradient with other MFS symporters. This research also focuses on the use of thirty-one camelid nanobodies to stabilize LacY in different intermediate states to be studied by X-ray diffraction. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate.