DE NOVO DESIGN OF FUNCTIONAL CHANNEL PROTEINS

通道蛋白是一类特殊的膜蛋白， 细胞信号传导是细胞内稳态的关键控制元件。 它们的功能障碍导致疾病过程， 药物干预的主要目标。 最终目的是了解 分子设计的基本原理 的 目前的目标是确定最低单位的发生率， 结构与特定的功能属性作为一个易于处理的方法， 研究序列结构决定论。 的新奇 战略在于基于以下的离散模块化组装的概念： 前提是小的、独立折叠的模块可以稳定地 整个通道蛋白的缺失和结构 确定在脂质双层中重构的这种分离的模块， 可行和现实。 具体来说，该计划侧重于电压- 门控通道蛋白 该战略认为，鉴于主要 通道蛋白的结构，它可能是可能的，以确定功能 与所述离子孔和所述电压传感器相关联的模块，以及 这些序列可以折叠成稳定的基序， 恢复渗透和选通特性， 完整的渠道。 序列分析和构象能 计算指导设计。 蛋白质是通过表达在 编码设计的通道蛋白的合成基因的细菌。 信道特性通过重构设计的 脂质双层中的蛋白质和通过cDNA的异源表达 在两栖动物卵母细胞中编码蛋白质。 离子电流 测量提供了渗透性能和渗透率的测定。 通道概率的电压依赖性调节 处于开放或封闭状态。 位移电流 测量探测状态之间的构象转变。 蛋白质结构是由多维核磁共振光谱确定的 同位素标记的蛋白质在氘代洗涤剂胶束和 定向磷脂双层膜中固体核磁共振 结构- 功能关系是由结构的收敛发展起来的， 信息与信道功能的表征。 位点特异 替代物有助于改进结构-功能图。 最终 一个成功的设计的测试是重述的功能属性， 将整个蛋白质从独立的模块组装起来。的 开发一套模块，通过组合和 排列可能产生功能多样性，是令人兴奋的和现实的。 这些发现可能有助于了解 并为药物设计提供结构蓝图。

Channel proteins, a special class of membrane proteins which mediate cell signaling, are pivotal control elements of cellular homeostasis. Their dysfunction leads to disease processes and they constitute a prime target for drug intervention. The ultimate goal is to understand the fundamental principles underlying their molecular design. The immediate objective is to establish the occurrence of minimum units of structure with specific functional attributes as a tractable approach to investigate the sequence-structure determinism. The novelty of the strategy resides in the notion of a discrete modular assembly based on the premise that small, independently folded modules may be stable in the absence of the entire channel protein and that structure determination of such isolated modules reconstituted in lipid bilayers is feasible and realistic. Specifically, the program is focused on voltage- gated channel proteins. The strategy considers that given the primary structure of channel proteins it may be possible to identify functional modules associated with the ionic pore and the voltage sensor, and that such sequences may fold predictably into stable motifs that will retrieve the permeation and gating properties which are characteristic of intact channels. Sequence analysis and conformational energy calculations guide the designs. Proteins are produced by expression in bacteria of synthetic genes encoding the designed channel proteins. Channel properties are established by reconstitution of designed proteins in lipid bilayers and by heterologous expression of cDNAs encoding the proteins in amphibian oocytes. Ionic current measurements provide an assay of the permeation properties and of the voltage-dependent regulation of the probability of the channel residing in the open or closed states. Displacement current measurements probe the conformational transitions between states. Protein structure is determine by multidimensional NMR spectroscopy of isotopically labeled proteins in deuterated detergent micelles and by solid-state NMR in oriented phospholipid bilayer lamellae. Structure- function relations are developed by the convergence of structural information with the characterization of channel function. Site-specific replacements assist in refining a structure-function map. The ultimate test of a successful design is recapitulation of functional attributes of the whole protein by assembling it from independent modules. The development of a repertoire of modules, that by combination and permutation may generate functional diversity, is exciting and realistic. These discoveries may contribute clues to understand mechanisms of disease and provide structural blueprints for drug design.