DE NOVO DESIGN OF FUNCTIONAL CHANNEL PROTEINS

该计划的最终目标是确定基本原则 这决定了通道蛋白的生物设计。最直接的 目标是实现成孔结构的分子设计和 用它来理解离子选择性的分子基础， 通道开放概率的通道阻塞和电压调节。这个 中心概念是，鉴于通道蛋白的一级结构，它 可能识别将可预测地折叠的功能模块 转化为稳定的结构主题，并实现了 正宗的系统。这一努力的第一步是只对大多数 离子通道功能的基本单位，即成孔 结构。关于成孔结构的合理的分子蓝图 是一束聚集在一起的两亲性α-螺旋 共同产生亲水性通道。这种孔结构是 由代表氨基酸序列的功能模块设计而成 真正的蛋白质和精炼以适应特定的功能 特点。下一层次的复杂性包括电压- 并考虑了最小电压门控通道的设计 它将表现出离子选择性的基本孔道性质 跨膜电位的额外调节。该方法 涉及：(1)基于以下公式的蛋白质结构模型的制定 序列分析和二级结构预测；(2)构象 评估拟议结构有效性的能量计算 基元，设计“计算机突变”来指导实验设计，以及 以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 固相合成多肽及其直接表达的结构 合成基因编码设计的通道蛋白；[4]功能 人工合成蛋白质重组对设计通道的分析 在平面脂质双分子膜中以及相应的cRNA在 两栖类卵母细胞或哺乳动物细胞中的cDNA.单通道电流 电压钳位条件下的录音提供了一组详细的 决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)结构功能评价中的选址置换 关系；[6]用多维核磁共振确定蛋白质结构 氚洗涤剂中同位素标记蛋白质的光谱研究 胶束和定向磷脂双层片层中的固体核磁共振。 预计结构性信息与 在单个水平上详细分析通道蛋白的功能 分子事件，结合了多肽合成和 在分子建模的指导下，重组DNA技术将提供 进行结构-功能图谱系统研究的途径 通道蛋白，并可能提供有关生物设计的线索 这类蛋白质是活细胞的基本成分。

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.