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-螺旋的从头蛋白质的文库 结构.这一目标将通过一种同时利用 合理设计和组合方法。第一步将需要 设计一种新的结构支架，其指定独特折叠的4-螺旋 捆。每个序列位置将被定义为出现在特定的 在所需的α-螺旋结构中的环境（暴露与掩埋; α-螺旋对转角等）。第二步将使用组合方法， 产生与设计的氨基酸序列一致的从头氨基酸序列的文库。 脚手架组合多样性将不是随机的，而是随机的。 设计为在每个位置仅递送那些最相容的氨基酸 与支架中该位置的结构环境相匹配。 本项目的具体目标是：（1）设计一种新型结构脚手架 指定4-螺旋束;（2）设计和构建一个文库， 编码大量蛋白质序列的合成基因一致 用这种新的支架;（3）表达和纯化从头蛋白， 收集;（4）生物药理学特征的结构和热力学 纯化的蛋白质的性质，从而评估它们是否形成熔融 小球系综或独特的折叠结构;和（5）以确定 3-代表性蛋白质的三维结构的NMR光谱。 设计和建造大量独特折叠的设计的能力。 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， 天然基因和蛋白质的修饰和生产，未来 应用将不限于自然界提供的大分子。的 产生良好折叠的从头蛋白文库的能力是一个初始的， 朝着发现新型蛋白质的最终目标迈出了重要的一步 在工业和医学上的应用。

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.