Chaperone Assisted Crystallography

描述(申请人提供)：我们建议开发一套强大而通用的方法，这将极大地扩展成功解决最具挑战性的一类大分子晶体结构(膜蛋白质、大分子复合体和功能RNA)的能力。我们的方法，伴侣辅助结晶学(CAC)，是基于利用“结晶伴侣”--设计的结合结构域，目标是结合到选定的分子实体--来促进结晶并提供晶相信息。这种方法已经被证明在结晶一些重要的膜蛋白方面非常成功，其中抗体片段在形成有效的晶格接触中起主导作用。这些成功表明，CAC策略可能广泛适用于各种结构生物学靶点，但它们依赖于传统的杂交瘤方法来生产抗体，这种方法繁琐、效率低下且成本高昂。为了绕过这些技术难题，我们将采用新型抗体组合文库、噬菌体和酵母展示技术的强大组合来生产伴侣分子。我们的初步结果表明，一个新的抗体组合文库，包括一个“简化的遗传密码”，即只有四种氨基酸类型，与包含所有20种氨基酸类型的抗体组合文库一样能产生与靶标的结合松弛。蛋白质工程的这一重大突破的重要性在于，它允许显著增加可以组合多样化的伴侣位点，正是这一特征有效地产生了高亲和力的伴侣蛋白。我们将使用基于不同分子支架的三个伴侣类别：抗体Fab结构域(约为 包括一个骆驼VHH结构域(约130个氨基酸)和一个纤维连接蛋白III型结构域(约90个氨基酸)。我们将针对不同的支架和不同的目标类别优化简化的遗传编码。我们将测试“伴侣辅助晶格引发剂”的新概念，我们将设计更高阶的伴侣组装，以实现高效的结晶。我们将以高吞吐量管道格式组织我们方法的不同技术模块。我们的技术易于并行化，其中大部分可以传播到小型实验室环境中，为各个研究小组提供强大的工具来解决主要的结构生物学问题。在这个项目中产生的伴侣本身也将是生物医学研究中非常有价值的工具，并可以取代杂交瘤产生的抗体。

DESCRIPTION (provided by applicant): We propose to develope a set of powerful and versatile methodologies which will greatly expand the capability to successfully solve the most challenging classes of macromolecule crystal structures (membrane proteins, macromolecular complexes and functional RNAs). Our approach, Chaperone Assisted Crystallography (CAC), is based on utilizing "crystallization chaperones"-engineered binding domains targeted to bind to a chosen molecular entity- to facilitate crystallization and provide crystallographic phasing information. This approach has been proven to be spectacularly successful in crystallizing a number of important membrane proteins, where antibody fragments play a dominant role in forming effective lattice contacts. These successes suggest that the CAC strategy may be broadly applicable to a wide variety of structural biology targets, but they relied on the classic hybridoma approach for antibody production, which is cumbersome, nherently inefficient and expensive. To circumvent these technical problems, we will employ a powerful combination of novel antibody combinatorial libraries, and phage and yeast display technologies to produce the chaperone molecules. Our preliminary results show that a novel antibody combinatorial library, comprising a "reduced genetic code"; i.e., only four amino acid types, is as powerful in producing binding laffinity to a target, as one that contains all 20 amino acid types. The importance of this major breakthrough in protein engineering is that it allows a significant increase in chaperone sites that can be combinatorially diversified, and it is this feature that efficiently produces high affinity chaperones. We will use three chaperone classes based on different molecular scaffolds: an antibody Fab domain (approximately 400 amino acids), a camelid VHH domain (approximately 130 aa), and a fibronectin type III domain (approximately 90 aa). We will optimize reduced genetic codes for different scaffolds and different target classes. We will test the novel concept of "chaperone assisted lattice initiators," where we will engineer higher order chaperone assemblies for efficient crystallization. We will organize distinct technology modules of our approach in a high throughput pipeline format. Our technology is amenable to parallelization, and much of it can be disseminated to a small lab environment, providing powerful tools to attack major structure biology problems to the individual research groups. The chaperones produced in this project themselves will also be highly valuable tools in biomedical research and could supercede the hybridoma-produced antibodies.