DE NOVO DESIGN OF FUNCTIONAL CHANNEL PROTEINS

通道蛋白--一类特殊的膜蛋白 细胞信号转导，是细胞内稳态的关键控制因素。 它们的功能障碍会导致疾病进程，它们构成了 毒品干预的首要目标。最终的目标是理解 它们分子设计的基本原理。这个 近期目标是确定发生的最小单位数 结构作为一种易于处理的方法来实现 研究序列结构决定论。它的新奇之处在于 战略存在于离散模块组装的概念中，该组件基于 小的、独立折叠的模块可能在 缺乏完整的通道蛋白和该结构 在脂质双层中重组的这种分离模块的测定是 切实可行，实事求是。具体地说，该计划专注于电压- 门控通道蛋白。该战略认为，鉴于主要的 通道蛋白的结构有可能鉴定出功能 与离子孔和电压传感器相关联的模块，以及 这样的序列可能会可预测地折叠成稳定的基序 检索具有特征的渗透和浇注属性 完整的航道。序列分析与构象能 计算是设计的指导。蛋白质是通过在体内表达而产生的 细菌合成编码所设计的通道蛋白的基因。 通道属性是通过对设计的 脂双层中的蛋白质与cDNA的异源表达 编码两栖类卵母细胞中的蛋白质。离子流 测量提供了渗透性能的分析和 信道概率的电压依赖性调节 处于开放或关闭状态的。位移电流 测量探测状态之间的构象转变。 蛋白质结构由多维核磁共振波谱确定 在氚洗涤剂胶束中的同位素标记的蛋白质 定向磷脂双层片层的固体核磁共振。结构- 函数关系是通过结构的收敛来发展的 具有信道功能表征的信息。特定于站点 替换有助于完善结构-功能图。终极的 成功设计的测试是对以下功能属性的概括 通过从独立的模块中组装完整的蛋白质。这个 开发一系列模块，通过组合和 排列可以产生功能多样性，是令人兴奋和现实的。 这些发现可能有助于理解 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.