NIRT: Combinatorial Engineering of Nanomachines: Building Novel Membrane Proteins via De Novo Design and Directed Evolution

本提案是响应NSF 01- 157号公告而收到的。本提案的总体目标是：a）基于复杂的计算化学技术与体外定向分子进化的整合，开发用于膜蛋白和蛋白质组装体的设计和工程化的新方法和工具; B）基于细菌孔蛋白OmpF设计新的膜孔，用于控制膜囊泡的渗透性; c）设计基于流感病毒血凝素的新型膜融合机器，用于调节双层融合和膜蛋白展示; d）进一步理解膜蛋白结构和活性的物理和化学性质; e）在这些跨学科的纳米科学研究方法中培训科学和工程专业的学生。4 nm厚度的脂质双层，可以介导酶功能或构象变化，需要在纳米水平上控制蛋白质结构（和亚纳米）尺度。在他们的建议中，新的膜蛋白将通过结合合理设计，通过组合库的部分随机设计和定向进化来构建。两个不同的系统将集中在作为出发点：细菌孔蛋白，这是大的渗透性膜孔，和流感病毒蛋白血凝素，这是一个pH诱导的膜融合。对于这两种系统，已经积累了大量的结构和功能数据，并且存在使用它们来构建有用的基于膜的装置的巨大潜力。具有改变和调节的渗透性的通道可用于选择性地将化合物递送至周围环境或选择性地内化和处理外部底物。类似地，门控融合子可用于控制含有两种不同反应物的囊泡之间的混合，并且还可被工程化以充当调节蛋白质结构域的展示的开关。门控孔和融合剂也可以被整合到合成脂质组装体中，以构建新的“智能”材料，其体积弹性和/或渗透性可以根据环境信号进行调节。除了他们将设计的蛋白质的特定用途之外，他们将开发的工具和出现的新膜蛋白质将为相对原始的膜蛋白质设计领域提供有价值的见解。可溶性蛋白质的工程化已经发展成为一个巨大的领域，它发展迅速，远远领先于膜蛋白的相应领域。特别是，定向进化的强大工具，它已经产生了广泛的新的可溶性蛋白质，还没有被应用到膜活性蛋白质的设计。

This proposal was received in response to the announcement NSF 01-157.The overall objectives of this proposal are to: a) develop novel methods and tools for design and engineering of membrane proteins and protein assemblies based upon the integration of sophisticated computational chemistry techniques with in vitro directed molecular evolution; b) engineer novel membrane pores based upon the bacterial porin OmpF for controlling membrane vesicle permeability; c) engineer novel membrane fusion machines based upon influenza virus hemagglutinin for regulating bilayer fusion and membrane protein display; d) further the understanding of the physical and chemical properties underlying membrane protein structure and activity; e) train science and engineering students in these interdisciplinary nanoscience research methods.To incorporate stable proteins that span the ~4nm thickness of the lipid bilayer and that can mediate enzymatic functions or changes in conformation requires control of protein structure at the nanometer (and sub-nanometer) scale. In their proposal, novel membrane proteins will be constructed by combining rational design, partially random design via combinatorial libraries, and directed evolution. Two different systems will be focused on as starting points: bacterial porins, which are large permeability membrane pores, and the influenza virus protein hemagglutinin, which is a pH inducible membrane fusogen. A great deal of structural and functional data has been accumulated for both of these systems, and enormous potential exists for using them to build useful membrane based devices. Channels with altered and regulated permeability could be used to selectively deliver compounds to the ambient environment or selectively internalize and process external substrates. Similarly, gated fusogens could be used to control mixing between vesicles containing two different reactants and could also be engineered to act as switches that regulate the display of protein domains. Both gated pores and fusogens could also be incorporated into synthetic lipid assemblies in order to construct new "smart" materials, whose bulk elasticity and/or permeability are modulated in response to environmental signals.Beyond the specific utility of the proteins that they will engineer, the tools that they willdevelop and the new membrane proteins that emerge will provide valuable insight into therelatively primitive field of membrane protein design. The engineering of soluble proteins hasburgeoned into an enormous field that is moving rapidly and is far ahead of the corresponding field for membrane proteins. In particular, the powerful tool of directed evolution, which has given rise to a wide range of new soluble proteins, has not been applied to the design of membrane-active proteins.