Self-Interacting Transmembrane Helices from the Human Single-Pass Membrane Proteome: The Impact of Primary Structure and Lipids on Affinity and Stoichiometry

来自人类单程膜蛋白质组的自相互作用跨膜螺旋：一级结构和脂质对亲和力和化学计量的影响

• 批准号: 105798956
• 负责人: Professor Dr. Dieter Langosch
• 金额: --
• 依托单位: Lehrstuhl für Chemie der Biopolymere
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/105798956?language=en
• 关键词: Self; Interacting; Transmembrane; Helices; Human

Single-pass membrane proteins comprise a functionally diverse set of proteins that corresponds to >10% (~2200 proteins) of the human proteome. Many of these proteins are already known to homomerize via transmembrane domains (TMDs). The underlying helix-helix interfaces are frequently built from complex motifs formed from different types of amino acids. It is currently not known how many different types of interfacial amino acid motifs exist and how they relate to the respective protein quaternary structure and function. Our recently published comprehensive bioinformatic analyses of the entire human single-pass membrane proteome that were connected to experimental assessments of TMD-TMD interactions can briefly be summarized as follows. First, based on TMD sequence homology, a significant fraction (13.5 %) of the TMDs can be grouped into clusters mostly consisting of paralogs. Some of these clusters are characterized by high-affinity TMD self-interaction not reported before. Second, hundreds of TMD helices exhibit non-random one-sided residue conservation that is useful in predicting correlated homotypic interactions.In this follow-up application, we will exploit these previous results. In Goal 1, we plan to apply scanning mutagenesis and the in vivo ToxR assay to comprehensively investigate the sequence specificity and the role of amino acid motifs in TMD-TMD interaction. Besides expanding the list of high-affinity TMDs, bioinformatic analyses of the results will reveal the connection between the evolutionary conservation of amino acids and their role in the helix-helix contacts. In Goal 2, we will develop a novel in vitro homoFRET assay to determine the stoichiometry of interaction for these TMDs, and investigate the influence of positively charged flanking residues and negatively charged lipids in homodimerization. By combining both in vivo and in vitro assays in this comparative study, we hope to make broad conclusions regarding some of the predominant questions in the field.

单通道膜蛋白包含一组功能多样的蛋白质，相当于人类蛋白质组的>10%（约2200种蛋白质）。已知这些蛋白质中的许多通过跨膜结构域（TMD）进行同源化。潜在的螺旋-螺旋界面通常由不同类型的氨基酸形成的复杂基序构建。目前尚不清楚有多少不同类型的界面氨基酸基序存在，以及它们如何与各自的蛋白质四级结构和功能相关。我们最近发表的全面的生物信息学分析，整个人类的单程膜蛋白质组，连接到实验评估TMD TMD的相互作用可以简要总结如下。首先，基于TMD序列同源性，可以将显著部分（13.5%）的TMD分成主要由旁系同源物组成的簇。其中一些簇的特征在于高亲和力的TMD自相互作用之前没有报道。其次，数百个TMD螺旋表现出非随机的单侧残基保守性，这在预测相关的同型相互作用中是有用的。在目标1中，我们计划应用扫描诱变和体内ToxR测定来全面研究序列特异性和氨基酸基序在TMD-TMD相互作用中的作用。除了扩大高亲和力TMD的名单，结果的生物信息学分析将揭示氨基酸的进化保守性和它们在螺旋-螺旋接触中的作用之间的联系。在目标2中，我们将开发一种新的体外homoFRET测定，以确定这些TMD相互作用的化学计量，并研究带正电荷的侧翼残基和带负电荷的脂质在同源二聚化中的影响。通过结合体内和体外试验在这项比较研究中，我们希望在该领域的一些主要问题，使广泛的结论。