Conformational Changes in Secondary Active Transporters

今年，我们扩大了这个项目，包括GltPh，谷氨酸转运蛋白EAAT家族的模型和一种新的蛋白质，vcINDY，琥珀酸转运蛋白，它与长寿和肥胖有关。理解转运蛋白发挥作用的基本机制是至关重要的，因为这些知识可能导致开发对这些蛋白质有活性的治疗剂。我们试图分析的方式，以详细了解其机制的动态运动的功能运输。我们的方法是分析从细菌中获得的模型转运蛋白的运输细节。它们可以大量表达和纯化，并且适用于它们的哺乳动物表亲所不具备的生物物理方法。 我们继续我们的工作，使用EPR光谱监测GltPh的构象变化。这项工作已经确定了蛋白质的局部变化，这可能是重要的驱动离子，Na+和底物，天冬氨酸之间的耦合。我们正在继续努力确定这一变化的性质。 我们最近报道，gltPH的细胞外环必须是完整的有效运输。今年，我们详细探索了这种效应的机制，发现当34环被切断时，蛋白质保持底物亲和力，但最大转运显著降低。我们证明了这种效应与底物易位步骤的活化能有关，暗示了易位结构域的活塞样运动中的环。今年我们还发现，只有底物结合形式的蛋白质的转运受到影响-载脂蛋白，无底物转运蛋白不受34环切割的影响。我们已经进行了重要的控制，消除了这些影响的替代解释，一份描述这项工作的文件正在审查中。 今年，我们还开始研究一种新的转运蛋白vcINDY，它对果蝇的长寿很重要，并与哺乳动物的肥胖和胰岛素抵抗有关。我们首次成功地对vcINDY进行了功能重建，并直接证明了它是一种Na+偶联琥珀酸转运蛋白。我们对底物进行了广泛的筛选，并确定该蛋白质每转运一个琥珀酸盐携带三个Na离子，它是产电的（但不具有未偶联的Cl-电导），并且它主要转运双电荷形式的琥珀酸盐。我们目前正在撰写这篇文章以供出版。

This year we expanded this project to include both GltPh, a model for the EAAT family of glutamate transporters and a new protein, vcINDY, a succinate transproter which has been implicated in longevity and obesity. It is critical to understand the fundamental mechanisms by which there transporters function because such knowledge could lead to the development of therapeutic agents active against these proteins. We seek to analyze the dynamic movements of the functioning transporter on the way to a detailed understanding of its mechanism. Our approach is to analyze the details of transport in model transporters obtained from bacteria. These can be expressed and purified in large quantities and are amenable to biophysical methods not available for their mammalian cousins. We have continued our work using EPR spectroscopy to monitor conformational changes in GltPh. This work has identified local changes in the protein that may be important for coupling between the driving ion, Na+, and the substrate, aspartate. We are continuing work to identify the nature of this change. We recently reported that a extracellular loop of gltPH must be intact for effective transport. This year we probed the mechanism of this effect in detail and found that when the 34 loop is cut the proteins maintains substrate affinities but maximal transport is significantly reduced. We demonstrated that this effect relates to the activation energy of the substrate translocation step, implicating the loop in the piston like movement of the translocation domain. This year we also found that only the translocation of the substrate-bound form of the protein is affected--the apo, substrate-free transporter is unaffected by 34 loop cleavage. We have performed important controls eliminating alternative explanations for these effects and a paper describing this work is under review. This year we also began work on a new transporter, vcINDY, which is important for longevity in flies and is involved in obesity and insulin resistance in mammals. We performed the first successful functional reconstitution of vcINDY and directly demonstrated that it is a Na+ coupled succinate transporter. We have performed an extensive screen for substrates and identified that the protein carries three Na ions per succinate transpoted, that it is electrogenic (but does not have an uncoupled Cl- conductance) and that it primarily transports the doubly charged form of succinate. We are currently writing this work up for publication.