Libraries of Uniquely Folded Alpha-Helical Proteins

描述：拟议研究的总体目标是设计和生产 折叠成有序的α-螺旋的从头蛋白质文库 结构。这一目标将通过使用两者的方法来实现 合理设计和组合方法。第一步将需要 一种新型折叠四螺旋结构脚手架的设计 捆绑。每个序列位置将被定义为出现在特定的 在所需的阿尔法螺旋结构中的环境(裸露与埋藏； Alpha-Helix与Turn等)。第二步将使用组合方法来 生成与设计的氨基酸序列一致的从头开始氨基酸序列文库 脚手架。组合多样性不会是随机的，而是随机的 设计成在每个位置只提供那些最相容的氨基酸 脚手架上那个位置的结构环境。 本项目的具体目标是：(1)设计一种新型的结构脚手架 指定了一个4-螺旋束；(2)设计和构建一个 合成基因编码大量一致的蛋白质序列 利用这种新的支架；(3)表达和纯化从头蛋白 收集；(4)生物物理表征结构和热力学 纯化的蛋白质的性质，从而评估它们是否形成熔融 球状整体或独特的折叠结构；以及(5)确定 用核磁共振波谱技术研究代表性蛋白质的三维结构。 能够设计和构建独特折叠的De的大型集合 Novo蛋白质将对生物技术和医学产生重大影响。 尽管目前生物技术的应用通常集中在控制上， 未来天然基因和蛋白质的修饰和生产 应用将不会局限于自然提供的大分子。这个 产生折叠良好的从头蛋白质文库的能力是一种初步的和 朝着发现新蛋白质的终极目标迈出的关键一步 Made“用于工业和医学应用。

DESCRIPTION: The overall goal of the proposed research is to design and produce libraries of de novo proteins that fold into well ordered alpha-helical structures. This goal will be pursued through an approach that uses both rational design and combinatorial methods. The first step will entail the design of a new structural scaffold specifying a uniquely folded 4-helix bundle. Each sequence position will be defined to occur in a particular environment in the desired alpha-helical structure (exposed vs. buried; alpha-helix vs. turn etc.). The second step will use combinatorial methods to generate a library of de novo amino acid sequences consistent with the designed scaffold. The combinatorial diversity will not be random, but instead, will be designed to deliver at each position only those amino acids most compatible with the structural environment of that position in the scaffold. The specific aims of this project are (1) to design a new structural scaffold that specifies a 4-helix bundle; (2) to design and construct a library of synthetic genes that encode a large collection of protein sequences consistent with this new scaffold; (3) to express and purify de novo proteins from this collection; (4) to biophysically characterize the structural and thermodynamic properties of the purified proteins and thereby assess whether they form molten globule ensembles or uniquely folded structures; and (5) to determine the 3-dimensional structures of representative proteins by NMR spectroscopy. The ability to design and construct large collections of uniquely folded de novo proteins will have a significant impact on biotechnology and medicine. Whereas current applications of biotechnology typically focus on the control, modification, and production of naturally occurring genes and proteins, future applications will not be limited to macromolecules provided by nature. The ability to produce libraries of well folded de novo proteins is an initial and essential step towards the ultimate goal of discovering novel proteins "tailor made" for applications in industry and medicine.