STRUCTURE FUNCTION AND REGULATION OF GAP JUNCTION PROTEINS

我们的中心目标是了解缝隙连接化学调控的分子基础及其 对细胞行为的影响。该项目自#年启动以来一直是方案项目赠款的一部分。 1990年。最近，我们已经证明了羧基之间的直接、依赖于pH的分子内结合。 Cx43的末端(CT)结构域和细胞质环的后半部分。我们提出了特定的多肽 可以被设计成干扰这种相互作用并调节Cx43的调节。我们使用了高吞吐量 噬菌体展示试验鉴定与Cx43CT结合的多肽。我们发现Cx43CT 优先与含有特定“RXP”基序的多肽结合。几种RXP多肽与Cx43CT结合 具有微摩尔亲和力，导致Cx43CT结构域的结构修饰和Cx43通道的修饰 功能与规范。这项建议的目标是进一步表征这些多肽并寻找 可以调制Cx43通道功能的类似序列。具体目标是：目标1。)至 表征了RXP/Cx43CT络合物的高阶结构。目标2。)为了描述RXP的影响- 1和RXP-4对Cx43通道的调节。目标3。)要找出调停 RXP多肽与Cx43CT的结合及其对Cx43通道的功能影响我们将使用迭代 光谱方法的组合(核磁共振、表面等离子体声)、 蛋白质组学方法(高通量偏向噬菌体展示；多肽合成)和功能分析 开发一种与具有更高亲和力和选择性功能效应的多肽相关的结构活性。 这些多肽有可能成为操纵Cx43通道及其调控的探针。这些 研究将有助于更好地理解连接蛋白在健康和疾病中的作用。

Our central goal is to understand the molecular basis for the chemical regulation of gap junctions and its implications on cell behavior. This project has been a part of the Program Project Grant since its inception in 1990. Recently, we have shown direct, pH-dependent intramolecular association between the carboxyl terminal (CT) domain and the second half of the cytoplasmic loop of Cx43. We propose that specific peptides can be designed to interfere with this interaction and modulate Cx43 regulation. We have used a highthroughput phage display assay to identify peptides that bind to Cx43CT. We found that Cx43CT preferentially bound to peptides containing a specific "RXP" motif. Several RXP peptides bind to Cx43CT with micromolar affinity, cause structural modifications in the Cx43CT domain and modify Cx43 channel function and regulation. It is the goal of this proposal to further characterize these peptides and search for analogous sequences which may modulate the function of Cx43 channels. The specific aims are: Aim 1.) To characterize the high-order structure of RXP/Cx43CT complexes. Aim 2.) To characterize the effects of RXP- 1 and RXP-4 on the regulation of Cx43 channels. Aim 3.) To identify the structural constraints mediating the binding of RXP peptides to Cx43CT and their functional effects on Cx43 channels. We will use an iterative combination of spectroscopic methods (Nuclear Magnetic Resonance, Surface Plasmon sonance), proteomics approaches (high-throughput biased phage display; peptide synthesis) and functional assays to develop a structural-activity correlate toward peptides with higher affinity and selective functional effects. These peptides will potentially be probes for the manipulation of Cx43 channels and their regulation. These studies will lead to a better understanding of the role of connexins in health and disease.