Crystallization-neurotransmitter transporter homolo(RMI)

描述（由申请人提供）：突触性神经传递需要精确控制神经递质的释放和再摄取。再摄取是由突触前质膜上的特定转运蛋白进行的。这些转运体从突触间隙重新摄取神经递质，从而使突触前和突触后受体失活。这些转运体包括Na+/ ci依赖的血清素转运体（SERT）、多巴胺转运体（DAT）和去甲肾上腺素转运体（NET），它们都是神经递质-钠同向转运体（NSS）家族的成员。除了在正常神经传递中发挥关键作用外，这些蛋白质还与广泛使用的抗抑郁药（如氟西汀（百忧解）、丙咪嗪和帕罗西汀）或精神兴奋剂（如可卡因、安非他明和摇头丸）直接相互作用，所有这些药物都能抑制神经递质的再摄取。尽管NSS蛋白具有重要的生理、药理学和临床意义，但由于缺乏过表达哺乳动物膜蛋白的系统，目前尚无任何NSS蛋白的高分辨率结构信息。为此，我们从7个选定的哺乳动物神经递质细菌同源物中过表达和纯化了5个，以毫克为单位。我们已经确定其中一种在许多洗涤剂中稳定且单分散，因此适合结晶。该蛋白与人类转运蛋白序列同源性为20-25%。我们建议培养这种细菌NSS蛋白的二维晶体，并使用冷冻电子显微镜在大约10 A的分辨率下确定其结构。这张图应该揭示了蛋白质的一般结构及其在膜中的低聚状态。我们还建议对蛋白质进行三维结晶，达到大约3 A的分辨率，晶体将在项目的下一阶段使用x射线晶体学来解决其高分辨率结构。

DESCRIPTION (provided by applicant): Synaptic neurotransmission requires precise control of the release and reuptake of neurotransmitters. The reuptake is carried out by specific transporter proteins from the presynaptic plasma membrane. These transporters reuptake the neurotransmitters from the synaptic cleft, thereby inactivating both pre- and postsynaptic receptors. The transporters include the Na+/CI-dependent, serotonin transporter (SERT), dopamine transporter (DAT) and norepinephrine transporter (NET), all members of the neurotransmitter- sodium symporter (NSS) family. In addition to their key role in normal neurotransmission, these proteins interact directly with the widely prescribed antidepressants (e.g. fluoxetine (Prozac), imipramine and paroxetine) or psychostimulants (e.g. cocaine, amphetamine and MDMA (ecstasy)), all of them inhibit the neurotransmitter reuptake. In spite of their critical physiological, pharmacological and clinical importance, no high-resolution structural information is available for any NSS protein, partially due to the lacking of systems of overexpressing mammalian membrane proteins. To this end, we overexpressed and purified 5 out of 7 selected bacterial homologues of the mammalian neurotransmitter transporters in milligram quantities. We have identified one of them to be stable and monodisperse in a number of detergents and, thus, suitable for crystallization. The protein shares 20-25% sequence identity with the human transporters. We propose to grow two-dimensional crystals of this bacterial NSS protein and to determine its structure at approximately 10 A resolution using cryo-electron microscopy. This map should reveal the general architecture of the protein and its oligomeric state in the membrane. We also propose to crystallize the protein in three-dimensions to approximately 3 A resolution, and the crystals will be used to solve its high resolution structure using X-ray crystallography in the next stage of the project.