MT COBRE: MAPPING SEROTONIN TRANSPORTER BINDING DOMAINS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 5-羟色胺转运体(SERT)是一种完整的膜蛋白，负责神经元放电后内源性神经递质5-羟色胺从突触间隙中清除。SERT结构-功能的异常和/或SERT受体密度的变化与多种重大的神经和精神健康疾病有关。因此，有必要加强对SERT运作的了解。SERT全球三级结构的确切细节以及与SERT运输现象有关的关键蛋白质结合区的三维(3D)表示仍未明确。SERT结构组织被认为具有一个由SERT三级蛋白质结构在三维空间组装的氨基酸组成的配体结合部位。我们的假设是，对SERT结合区氨基酸组成的有效定位将揭示SERT三级结构的关键特征。我们的长期目标是在结构-功能方面显著促进对关键中枢神经系统转运蛋白之间的共性和差异的理解。这个项目的最初目标是用定制的、有效的和选择性的、基于奎帕嗪的交联探针来定位关键SERT结合域中的参与氨基酸残基。实现以下三个具体目标： 1.建立能够准确测量残基间二级结构的SERT三维计算模型。 2.合成一系列由我们的计算衍生配体设计标准定义的单功能和同双功能探针，并对其进行药理学表征。 3.确定氨基酸交联的SERT三维结构图，首先通过双头交联剂的不可逆化学标记来表达SERT，然后进行质谱标记的SERT序列分析，然后将标记的残基与3D SERT模型进行关联。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The serotonin transporter (SERT) is an integral membrane protein responsible for clearance of the endogenous biogenic neurotransmitter 5-HT from the synaptic cleft after neuronal firing. Abnormalities in SERT structure-function and/or changes in SERT receptor densities have been implicated in a variety of significant neurological and mental health disorders. Thus, an enhanced understanding of SERT operation is needed. The exact details of SERT global tertiary structure and intimate three-dimensional (3D) representations of critical protein binding regions linked to the SERT transport phenomena remain ill defined. The SERT structure organization is thought to possess a ligand binding site that is composed by amino acids assembled in 3D space by the SERT tertiary protein structure. It is our hypothesis that the efficacious mapping of the amino acid constituents of the SERT binding domains will reveal critical features of the SERT tertiary structure. Our long-term objective is to significantly contribute to understanding of the commonalities and differences amongst key CNS transport proteins in terms of structure-function. The original goal of this project has been to map participating amino acid residues within critical SERT binding domains with custom designed, potent and selective, quipazine-based cross-linking probes. The following three specific aims will be accomplished: 1. To construct a 3D computational model of SERT that will enable accurate measurements of inter-residue secondary structure. 2. To synthesize and pharmacologically characterize a collection of mono- and homo-bifunctional probes defined by our computationally derived ligand design criteria. 3. To determine the amino acid cross-linked SERT 3D structure maps, first by the irreversible chemical tagging of the dual-headed cross-linking agents to expressed SERT, followed by mass spectrometry tagged SERT sequence analysis, and then correlating the labeled residue sites with the 3D SERT model.