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

单通道膜蛋白包括一组功能不同的蛋白质，相当于人类蛋白质组的10%(~2200个蛋白质)。这些蛋白质中的许多已知通过跨膜结构域(TMD)同构化。潜在的螺旋-螺旋界面通常是由不同类型的氨基酸形成的复杂基序组成的。目前还不知道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。我们最近发表的对整个人类单程膜蛋白质组的全面生物信息学分析与TMD-TMD相互作用的实验评估有关，可以简要总结如下。首先，根据TMD序列的同源性，相当一部分(13.5%)的TMD可以归类为主要由平行序列组成的簇。其中一些星团具有以前没有报道过的高亲和力TMD自我相互作用的特征。第二，数以百计的TMD螺旋表现出非随机的单侧残基守恒，这对预测相关的同型相互作用很有用。在这个后续的应用中，我们将利用这些先前的结果。在目标1中，我们计划应用扫描突变和体内ToxR试验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的名单外，对结果的生物信息学分析将揭示氨基酸的进化保守与它们在螺旋-螺旋接触中的作用之间的联系。在目标2中，我们将建立一种新的体外HomoFRET实验来确定这些TMD相互作用的化学计量比，并研究正电荷侧翼残基和负电荷脂质在同质二聚中的影响。在这项比较研究中，通过结合体内和体外分析，我们希望对该领域的一些主要问题得出广泛的结论。

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.