ROLE OF ORDERED HELICAL SEGMENTS IN MEMBRANE PROTEINS

明确定义的螺旋多肽片段在 膜蛋白的组装和活性。这一点尤为明显。 在跨膜两亲性螺旋结合的通道蛋白中 与脂双层一起、插入其中并在其内齐聚以形成脂双层 生物活性结构。我们假设这些渠道的形成 活动在很大程度上是由独特的结构相互作用产生的 两亲性螺旋中的元素：一种疏水性--脂类相互作用 一个亲水成孔面和两个不对称的小氨基 含酸--螺旋-螺旋相互作用面。拟议的研究 将第一次描述其中每一项的贡献 两亲性螺旋面在组装和活动中的理想化 渠道毛孔。将特别强调对螺旋的定义- 螺旋接触产生二聚体以及更高形式的 齐聚作用。我们还将研究异质原子的性质。 螺旋的寡聚体组装，如在 乙酰胆碱受体通道与人囊性纤维化CFTRCl- 关于离子选择性和通道的开启和关闭 渠道毛孔。 这项工作将通过结合两种强大的技术来完成： 固相法多肽合成及生物物理分析 电生理测量，2D-核磁共振(溶液和固相)和 电子自旋共振(ESR)。合成肽和模板组装 螺旋束将被设计，合成，化学表征 然后在模型膜上进行研究。生物物理方法将绘制出 周围环境每个残留物、脂类外露、内衬通道或 参与螺旋-螺旋界面，以及确定 组装的孔道的齐聚数和通道活性。这个 这些研究的结果将有助于理解 膜蛋白组装和通道活性。稳定的一代 具有高离子选择性的模型通道-孔结构可以 宣扬这种结构作为药剂或在 生物医学设备。

Well defined helical polypeptide segments play a critical role in the assembly and activity of membrane proteins. This is particularly evident in channel proteins where transmembrane amphipathic helices associate with, insert into, and oligomerize within, the lipid bilayer to form bioactive structures. We hypothesize that these channel-forming activities arise, largely, from the interaction of the unique structural elements within amphipathic helices: a hydrophobic-- lipid interacting face, a hydrophilic--pore forming face and two asymmetric, small amino acid containing -- helix-helix interacting faces. The proposed research will describe, for the first time, the contributions each of these amphipathic helix faces play in the assembly and activity of idealized channel pores. Particular emphasis will be placed on defining the helix- helix contacts that give rise to dimers as well as higher forms of oligomerization. We will also examine the properties of hetero- oligomeric assemblies of helices, such as those proposed in the acetylcholine receptor channel and the human cystic fibrosis CFTR Cl- channel, with respect to ion selectivity and the opening and closing of channel pores. This work will be accomplished by combining two powerful techniques: solid phase peptide synthesis and biophysical analysis employing electrophysiological measurements, 2D-NMR (solution and solid phase) and Electron Spin Resonance (ESR). Synthetic peptides and template assemble helical bundles will be designed, synthesized, chemically characterized and then studied in model membranes. The biophysical methods will map the environment around each residue, lipid exposed, lining the channel or involved in the helix-helix interface as well as determine the oligomerization number and channel activity of the assembled pores. The results obtained from these studies will aid in the understanding of membrane protein assembly and channel activity. The generation of stable model channel-pore structures with high degrees of ion selectivity could herald the uses of such structures as pharmacological agents or in biomedical devices.