DE NOVO DESIGN OF FUNCTIONAL CHANNEL PROTEINS

功能通道蛋白的从头设计

• 批准号: 3308873
• 负责人: MAURICIO S MONTAL
• 金额: $ 17.98万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-10 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3308873
• 关键词: Xenopus; Xenopus oocyte; biophysics; calcium channel; complementary RNA; computer simulation; conformation; glycine receptors; lipid bilayer membrane; membrane channels; membrane potentials; nicotinic receptors; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; physical model; protein engineering; protein folding; protein reconstitution; protein sequence; protein structure function; recombinant DNA; site directed mutagenesis; structural biology; thermodynamics; voltage gated channel

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.

该计划的最终目标是确定基本原则 决定通道蛋白的生物学设计。即时的 目标是实现成孔结构的分子设计并 用它来理解离子选择性的分子基础， 通道封锁和通道开放概率的电压调节。这 中心思想是鉴于通道蛋白的一级结构 或许可以识别出可预测折叠的功能模块 成稳定的结构基序并实现功能属性 正宗的系统。这项努力的第一步是仅对最 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，它们聚集在一起 一起形成亲水通道。这种孔隙结构是 由代表氨基酸序列的功能模块设计 真实的蛋白质并经过精炼以适应特定的功能 特征。下一个层次的复杂性包括电压 传感装置并考虑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隙特性 跨膜电位的额外调节。方法 涉及：（1）基于蛋白质结构模型的制定 序列分析和二级结构预测； (2)构象 能量计算以评估拟议结构的有效性 主题，设计“计算机突变”来指导实验设计，以及 获得离子能量分布的定量描述 通过设计的渠道进行扩散； [3] 设计合成 通过固相肽合成和直接表达的结构 编码设计的通道蛋白的合成基因； [4] 功能性 通过重建合成蛋白质来分析设计的通道 在平面脂质双层中并通过在中表达相应的cRNA 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、药理学的功能参数 通道开放概率的特异性和电压依赖性调节； (5) 结构功能评估的位点选择性替换 关系； [6] 多维核磁共振测定蛋白质结构 氘代洗涤剂中同位素标记蛋白质的光谱 胶束和定向磷脂双层片中的固态核磁共振。 预计结构信息与 单通道蛋白功能的详细分析 分子事件，与肽合成的优点相结合 以分子模型为指导的重组 DNA 技术将提供 系统研究结构-功能图谱的路径 通道蛋白，并可能提供有关其生物设计的线索 这类蛋白质是活细胞的基本组成部分。