DE NOVO DESIGN OF FUNCTIONAL CHANNEL PROTEINS

该计划的最终目标是确定基本原则 决定了通道蛋白的生物学设计。立即 目的是实现成孔结构的分子设计， 用它来理解离子选择性的分子基础， 通道阻断和通道开放概率的电压调节。的 核心概念是，给定通道蛋白的一级结构， 可以识别将可预测地折叠的功能模块 转化为稳定的结构基序，并实现 真实系统这一奋进的第一步是只建模最 离子通道功能的基本单位，即成孔 结构成孔结构的合理分子蓝图 通道蛋白是一束两亲性α螺旋， 一起产生亲水通道。这样的孔结构是 由代表以下氨基酸序列的功能模块设计： 真正的蛋白质和精制，以适应特定的功能 特色下一级的复杂性包括电压- 传感器件，并考虑了最小电压门控通道的设计 这将表现出离子选择性的基本孔特性， 通过跨膜电位的额外调节。的方法 包括：（1）基于以下公式制定蛋白质的结构模型： 序列分析和二级结构预测;（2）构象分析 能量计算，以评估拟议结构的有效性 基序，设计“计算机突变”来指导实验设计， 为了获得离子的能量分布的定量描述， 通过设计的通道扩散; [3]设计的合成 通过固相肽合成和通过直接表达 编码设计通道蛋白的合成基因; [4]功能 通过合成蛋白质的重构分析设计的通道 在平面脂质双层中，并通过在 两栖类卵母细胞或哺乳动物细胞中的cDNA。单通道电流 电压钳条件下的记录提供了一组详细的 确定离子选择性的功能参数，药理学 通道开放概率的特异性和电压依赖性调节; (5]用于结构-功能评价的位点选择性替换 关系; [6]多维核磁共振测定蛋白质结构 氘代去污剂中同位素标记蛋白质的光谱学 胶束和固态NMR在定向磷脂双层薄层。 预计结构信息与 详细分析了通道蛋白在单通道水平上的功能 分子事件，结合肽合成的益处， 在分子建模的指导下，重组DNA技术将提供 的结构-功能图的系统研究的路径 通道蛋白，并可能提供线索的生物设计， 这类蛋白质是活细胞的基本组成部分。

The ultimate goal of the program is to identify the fundamental principles that determine the biological design of channel proteins. The immediate goal is to realize the molecular design of a pore-forming structure and to use it towards understanding the molecular basis of ionic selectivity, channel blockade and voltage regulation of channel open probability. The central notion is that given the primary structure of channel proteins it may be possible to identify functional modules that will fold predictably into stable structural motifs and fulfill functional attributes of the authentic system. A first step in this endeavor is to model only the most fundamental unit of function of ion channels, namely the pore-forming structure. A plausible molecular blueprint for the pore-forming structure of channel proteins is a bundle of amphipathic alpha-helices that cluster together to generate a hydrophilic channel. Such pore structures are designed from functional modules that represent the amino acid sequence of authentic proteins and refined to accommodate specific functional characteristics. The next level of complexity incorporates the voltage- sensing device and considers the design of a minimum voltage-gated channel that would exhibit the essential pore properties of ionic selectivity with the additional regulation by transmembrane potential. The approach involves: (1) Formulation of a structural model of the protein based on sequence analysis and secondary structure predictions; (2) Conformational energy calculations to assess the validity of the proposed structural motifs, to design "computer mutations" to guide experimental design, and to obtain quantitative descriptions of the energy profile for ionic diffusion through the designed channels; [3] Synthesis of the designed structures by solid-phase peptide synthesis and by direct expression of synthetic genes encoding the designed channel proteins; [4] Functional analysis of the designed channels by reconstitution of synthetic proteins in planar lipid bilayers and by expression of corresponding cRNA in amphibian oocytes or cDNA in mammalian cells. Single channel current recordings under voltage-clamp conditions provide a detailed set of functional parameters to determine ionic selectivity, pharmacological specificity and voltage-dependent regulation of channel open probability; (5] Site-selective replacements for evaluation of structure-function relationships; [6] Protein structure determination by multidimensional NMR spectroscopy of isotopically labeled proteins in deuterated detergent micelles and by solid-state NMR in oriented phospholipid bilayer lamellae. It is anticipated that the convergence of structural information with the detailed analysis of channel protein function at the level of single molecular events, integrated with the benefits of peptide synthesis and recombinant DNA techniques, guided by molecular modeling, will provide paths for a systematic investigation of the structure-function map of channel proteins, and may provide clues about the biological design of this class of proteins that are fundamental components of living cells.