Exploring the structural and mechanistic basis of H+, Na+, and K+ coupling in secondary active glutamate transporters

离子耦合是二次活性转运蛋白的基本性质。离子耦合的结构和机制基础还不是很清楚，这是这一提议的主要主题。谷氨酸转运体非常适合于研究离子偶联，原因有两个。首先，古生物转运蛋白Gltph的晶体结构是已知的，并为探索提供了一个框架。其次，谷氨酸转运体家族的不同成员使用不同的偶联离子和底物：偶联离子化学计量。这种多样性将有助于确定钠离子、质子和钾离子选择性的结构基础。将在结构指导下将Na+偶联转运体Gltph转化为H+和K+偶联蛋白。首先，具有不同离子选择性的转运体之间将交换大的结构域，随后嵌合体的序列将被微调。这项工作将导致解开离子耦合的序列决定因素。同时，嵌合蛋白将被纳入结晶实验，这将为离子偶联提供结构基础。一个附带项目将集中在Gltph中钠和天冬氨酸结合的时间分辨机制。将EPR、单分子FRET和停流测量与不同的结晶实验相结合，可以解释输运循环，并得到一个改进的函数模型。总而言之，这项工作将提供对谷氨酸转运蛋白这一医学上重要的家族的基本见解，更具体地说，将提供关于次级转运蛋白中阳离子偶联的结构基础的基本见解。

Ion coupling is a fundamental property of secondary active transporters. The structural and mechanistic basis of ion coupling is not well understood, and is the main topic of this proposal. Glutamate transporters are exquisitely suited to study ion coupling for two reasons. First, crystal structures of the archaeal transporter Gltph are known and provide a framework for the exploration. Second, different members of the glutamate transporter family use different coupling ions, and substrate:coupling ion stoichiometries. The diversity will facilitate the determination of the structural basis of sodium ion, proton and potassium ion selectivity. A structure-guided conversion of the Na+ coupled transporter Gltph into H+ and K+ coupled proteins will be pursued. First large domains will be exchanged between transporters with different ion selectivity, and subsequently the sequences of the chimeras will be fine-tuned. The work will lead to the unraveling of the sequence determinants for ion coupling. In parallel, the chimeric proteins will be included in crystallization experiments, which will provide the structural basis of ion coupling. A side project will concentrate on the time resolved mechanisms of sodium and aspartate binding in Gltph. By combining EPR, single molecule FRET and stopped flow measurements with different crystallization trial the transport cycle will be explained and an improved functional model will result. In summary, the work will provide basic insights in the medically important family of glutamate transporters, and more specifically in the structural basis of cation coupling in secondary transporters.