Chaperone Assisted Crystallography

描述（由申请人提供）：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大分子晶体结构（膜蛋白，大分子复合物和功能rna）的能力。我们的方法，伴侣辅助晶体学（CAC），是基于利用“结晶伴侣”-工程结合域靶向结合选定的分子实体-促进结晶和提供晶体相位信息。这种方法已被证明在结晶许多重要的膜蛋白方面取得了惊人的成功，其中抗体片段在形成有效的晶格接触方面起着主导作用。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，这是繁琐的，固有的低效和昂贵的。为了规避这些技术问题，我们将采用新型抗体组合文库的强大组合，以及噬菌体和酵母展示技术来生产伴侣分子。我们的初步结果表明，一个新的抗体组合文库，包括一个“减少的遗传密码”；也就是说，只有四种氨基酸类型，在产生与目标的结合亲和力方面与包含所有20种氨基酸类型的氨基酸一样强大。这一重大突破在蛋白质工程中的重要性在于，它允许伴侣蛋白位点的显著增加，这些位点可以组合多样化，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种伴侣类：抗体Fab结构域(约

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.