Structure and Function in NSS transporters

描述（由申请人提供）：神经递质钠同向转运体（NSS）家族包括负责神经递质GABA、甘氨酸、去甲肾上腺素、5-羟色胺和多巴胺再摄取的转运体。它们代表神经传递中的关键元素，从而将神经递质的细胞外浓度调节在低水平，并且递质被有效地再循环。在这个家族中还有许多不参与神经传递的氨基酸转运蛋白，包括许多预测的功能尚未确定的原核蛋白。理解这些蛋白质的一个主要限制是难以表达和纯化大量蛋白质用于结构和功能研究。最近的证明，1原核NSS序列编码的功能性色氨酸转运蛋白（TnaT）开辟了新的途径，了解所有这些转运蛋白的结构和功能。该建议旨在利用这些新方法来了解TnaT，并将这些发现与哺乳动物神经递质转运蛋白联系起来。拟议的实验包括钠耦合色氨酸运输的生物能量学检查。这些实验将确定哪些离子梯度与色氨酸转运相结合，并将建立转运的化学计量。该提案还描述了使用遗传选择方法来估计蛋白质结构不同区域之间的物理接近度。最后，有实验来评估TnaT作为同源寡聚体发挥作用的可能性，并使用二维（2D）晶体学获得关于转运蛋白的结构信息。

DESCRIPTION (provided by applicant): The neurotransmitter sodium symporter (NSS) family includes transporters responsible for the re-uptake of neurotransmitters GABA, glycine, norepinephrine, serotonin, and dopamine. They represent a key element in neurotransmission whereby the extracellular concentration of neurotransmitter is regulated at a low level, and transmitters are efficiently recycled. Also in this family are many transporters for amino acids not involved in neurotransmission, including many predicted prokaryotic proteins whose function has not yet been identified. A major limitation in understanding these proteins has been the difficulty in expressing and purifying significant amounts of protein for structural and functional studies. The recent demonstration that 1 of the prokaryotic NSS sequences encodes a functional tryptophan transporter (TnaT) has opened up new avenues for understanding the structure and function of all of these transporters. This proposal is designed to take advantage of these new approaches to understand TnaT and to relate these findings to mammalian neurotransmitter transporters. The proposed experiments include an examination of the bioenergetics of sodium-coupled tryptophan transport. These experiments will determine which ion gradients are coupled to tryptophan transport and will establish the stoichiometry of transport. The proposal also describes the use of genetic selection methods to estimate the physical proximity between different regions of the protein structure. Finally, there are experiments to evaluate the possibility that TnaT functions as a homo-oligomer and to gain structural information about the transporter using two-dimensional (2D) crystallography.