MT COBRE: MAPPING SEROTONIN TRANSPORTER BINDING DOMAINS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。血清素转运体（SERT）是一种完整的膜蛋白，负责清除神经元放电后突触间隙中的内源性神经递质5-HT。SERT结构-功能异常和/或SERT受体密度变化与多种显著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全局三级结构的确切细节和与SERT转运现象相关的关键蛋白质结合区域的密切三维（3D）表示仍然不清楚。SERT结构组织被认为具有配体结合位点，该位点由SERT三级蛋白结构在三维空间中组装的氨基酸组成。我们的假设是，SERT结合域的氨基酸成分的有效映射将揭示SERT三级结构的关键特征。我们的长期目标是为理解关键中枢神经系统转运蛋白在结构功能方面的共性和差异做出重大贡献。该项目的目标是利用定制设计的、有效的、选择性的、基于喹帕嗪的交联探针来绘制关键SERT结合域内参与的氨基酸残基。将实现以下三个具体目标：1。构建SERT三维计算模型，实现残馀间二级结构的精确测量。2. 通过计算得出的配体设计标准，合成和药理学表征一系列单功能和同源双功能探针。3. 为了确定氨基酸交联SERT的三维结构图，首先通过不可逆化学标记双头交联剂来表达SERT，然后进行质谱标记SERT序列分析，然后将标记的残基位点与SERT三维模型进行关联。

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 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 goal of this project is 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.