Crystallization-neurotransmitter transporter homolo(RMI)

描述(申请人提供)：突触神经传递需要精确控制神经递质的释放和重新摄取。这种重摄取是由突触前质膜上的特定转运蛋白完成的。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。转运体包括钠/氯依赖转运体、5-羟色胺转运体(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-钠转运体(NSS)家族的成员。除了它们在正常神经传递中的关键作用外，这些蛋白还直接与广泛使用的抗抑郁药(如氟西汀(Prozac)、丙咪嗪和帕罗西汀)或精神刺激剂(如可卡因、安非他明和摇头丸)相互作用，所有这些都抑制神经递质的再摄取。尽管NSS蛋白具有重要的生理、药理和临床意义，但目前尚无任何NSS蛋白的高分辨结构信息，部分原因是缺乏哺乳动物膜蛋白的高表达系统。为此，我们超量表达并纯化了7个哺乳动物神经递质转运蛋白的7个细菌同源物中的5个。我们已经确定其中一种是稳定的，单分散在一些洗涤剂中，因此适合结晶。该蛋白质与人类转运蛋白有20%-25%的序列同源性。我们建议生长这种细菌NSS蛋白的二维晶体，并使用冷冻电子显微镜以大约10A的分辨率确定其结构。这张图应该揭示蛋白质的总体结构和它在膜中的低聚状态。我们还建议将蛋白质在三维空间中结晶到大约3A的分辨率，这些晶体将在项目的下一阶段使用X射线结晶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.