DE NOVO DESIGN OF FUNCTIONAL CHANNEL PROTEINS

功能通道蛋白的从头设计

基本信息

批准号：
6018960
负责人：
MAURICIO S MONTAL
金额：
$ 25.89万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-08-10 至 2001-07-31
项目状态：
已结题

项目摘要

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.

通道蛋白，一类特殊的膜蛋白，介导细胞信号传导是细胞稳态的关键控制元件。它们的功能障碍会导致疾病过程，并且它们构成了药物干预的首要目标。最终目标是了解其分子设计的基本原理。这近期目标是确定出现的最小单位具有特定功能属性的结构作为一种易于处理的方法研究序列结构决定论。的新颖性策略在于基于离散模块化组件的概念前提是小型的、独立折叠的模块可以稳定地存在整个通道蛋白及其结构的缺失脂质双层中重构的此类分离模块的测定是可行且现实。具体来说，该计划的重点是电压门控通道蛋白。该策略认为，鉴于主要通道蛋白的结构有可能识别功能与离子孔和电压传感器相关的模块，以及这样的序列可以预测地折叠成稳定的基序检索特有的渗透和门控特性完整的通道。序列分析和构象能计算指导设计。蛋白质通过表达产生编码设计的通道蛋白的合成基因的细菌。通道属性是通过重构设计来建立的脂质双层中的蛋白质和 cDNA 的异源表达编码两栖动物卵母细胞中的蛋白质。离子电流测量提供了渗透特性和通道概率的电压相关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。通过多维核磁共振波谱确定蛋白质结构氘化洗涤剂胶束中同位素标记的蛋白质的研究定向磷脂双层片中的固态核磁共振。结构- 功能关系是通过结构的收敛而发展起来的具有通道功能表征的信息。特定地点替换有助于完善结构功能图。终极成功设计的测试是功能属性的概括通过从独立模块组装整个蛋白质。这开发一系列模块，通过组合和排列可能产生功能多样性，令人兴奋且现实。这些发现可能为理解机制提供线索疾病并为药物设计提供结构蓝图。