MT COBRE: MAPPING SEROTONIN TRANSPORTER BINDING DOMAINS

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 5-羟色胺转运体（SERT）是一种膜蛋白，在神经元放电后负责从突触间隙清除内源性生物神经递质5-HT。SERT结构-功能的异常和/或SERT受体密度的变化与多种重要的神经和精神健康障碍有关。因此，需要增强对SERT操作的理解。SERT全球三级结构的确切细节和紧密的三维（3D）表示的关键蛋白质结合区域的SERT运输现象仍然不明确。 SERT结构组织被认为具有由通过SERT三级蛋白质结构在3D空间中组装的氨基酸组成的配体结合位点。我们假设SERT结合域的氨基酸组分的有效映射将揭示SERT三级结构的关键特征。我们的长期目标是在结构-功能方面为理解关键CNS转运蛋白之间的共性和差异做出重大贡献。 该项目的最初目标是用定制设计的、有效的和选择性的、基于喹帕嗪的交联探针绘制关键SERT结合结构域内的参与氨基酸残基。将实现以下三个具体目标： 1.构建SERT的3D计算模型，以实现残基间二级结构的精确测量。 2.合成并表征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源双功能探针。 3.为了确定氨基酸交联的SERT的3D结构图，首先通过双头交联剂对表达的SERT进行不可逆化学标记，然后通过质谱标记的SERT序列分析，然后将标记的残基位点与3D SERT模型相关联。

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.