Chaperone Assisted Crystallography

描述（由申请人提供）：我们已经开发并验证了一种强大的高通量方法，称为“伴侣辅助结晶学”（CAC），可以极大地促进解决最具挑战性的结构生物学问题。长期目标是将CAC平台发展到一个水平，使其应用程序通过使困难的问题成为常规和“不可能”的问题变得可行，从而对结构生物学社区产生变革性影响。CAC方法学基于使用合成衍生的抗体片段作为“结晶伴侣”，其特异性且紧密地结合靶蛋白或RNA实体，从而促进结晶并提供定相信息。形成CAC方法基础的技术突破是一种创新的组合文库设计，该设计采用“简化的遗传密码”来产生针对非常广泛的靶分子的高功能合成抗体片段。使用这种方法，我们已经产生了分子伴侣，包括膜蛋白和功能性RNA，促进结晶和随后的结构测定。主要成果包括全长KcsA钾离子通道和第1组内含子的P4 P6结构域的结构。为了进一步扩展CAC技术的功能，我们建议开发一些额外的增强功能。第二代CAC平台将包括：i）分别为膜蛋白和核酸定制的新的“简化遗传密码”分子伴侣文库; ii）捕获靶分子的优选构象状态; iii）将分子伴侣靶向靶分子上的特定预定义区域; iv）产生分子伴侣以瞬时大分子复合物（例如DNA-蛋白质相互作用）并稳定这种复合物的能力。V）Fab结合蛋白形式的“共分子伴侣”，其可被工程化以引入用于MAD定相的异常原子类型并改变表面性质，以及诱导晶格形成。新的CAC增强功能的有效性将根据一组五个高影响力的结构性问题进行评估，这些问题已被证明是使用传统方法完全无法解决的。这些“模型系统”包括：1）HIV 1整合酶-DNA复合物，2）F-肌动蛋白四聚体，3）MerR家族的转录因子，4）NaChBac Na+通道，和5）构象锁定形式的22-肾上腺素受体。从这些系统中获得的经验将用于进一步完善CAC技术。为了使这种强大的技术可用于结构生物学社区，我们已经建立了一个校外计划，研究人员可以将他们的柠檬酸蛋白发送给我们，通过CAC管道，或派遣人员到我们的实验室监督生产结晶分子伴侣。公共卫生相关性：我们正在开发伴侣辅助结晶（CAC）技术，以解决具有重要生物医学意义的蛋白质系统的结构，这些蛋白质系统通过传统方法无法结晶。一个关键的创新是我们能够快速生成设计抗体，这些抗体可以紧密结合靶分子，并促进高质量晶体的形成，用于X射线晶体学分析。该技术将对结构生物学社区产生广泛的影响，用它确定的结构将促进对细胞功能的基本理解，并为各种疾病的药物开发提供指导。

DESCRIPTION (provided by applicant): We have developed and validated a powerful high throughput approach called "Chaperone-Assisted Crystallography" (CAC) that can greatly facilitate solving the most challenging types of structural biology problems. The long-range goal is to develop the CAC platform to a level where its application will have a transforming effect on the structural biology community by making difficult problems routine and "impossible" problems feasible. The CAC methodology is based on the use of synthetically derived antibody fragments as "crystallization chaperones" that specifically and tightly bind to a target protein or RNA entity and thereby promote crystallization and provide phasing information. The technical breakthrough that forms the foundation of the CAC method is an innovative combinatorial library design that employs a "reduced genetic code" to produce highly functional synthetic antibody fragments to an extraordinary broad spectrum of target molecules. Using this approach we have produced chaperones to recalcitrant targets, including membrane proteins and functional RNAs, facilitating crystallization and subsequent structure determination. Major achievements include the structures of the full-length KcsA potassium ion channel and the P4P6 domain of group 1 intron. To further expand the capabilities of the CAC technology, we propose to develop a number of additional enhancements. The 2nd Generation CAC platform will include: i) new "reduced genetic code" chaperone libraries tailored for membrane proteins and nucleic acids, respectively; ii) trapping preferred conformational states of the targeted molecules; iii) targeting chaperones to a specific, predefined region on the target molecule; iv) the ability to produce chaperones to transient macromolecular complexes (e.g. DNA-protein interactions) and stabilize such complexes. v) "co-chaperones" in the form of Fab-binding proteins that can be engineered to introduce anomalous atom types for MAD phasing and alter surface properties, as well as to induce lattice formation. The effectiveness of the new CAC enhancements will be evaluated against a set of five high-impact structural problems that have proven to be totally intractable using traditional approaches. These "model systems" include: 1) HIV 1 Integrase-DNA complex, 2) F-actin tetramer, 3) MerR family of transcription factors, 4) NaChBac Na+ channel, and 5) conformationally locked forms of the 22-adregenic receptor. Experiences gained from working with these sytems will be used to further refine the CAC technology. To make this powerful technology available to the structural biology community, we have established an extramural program where investigators can send us their recalcitrant proteins to be put through the CAC pipeline, or send personnel to our labs to be supervised on producing crystallization chaperones. PUBLIC HEALTH RELEVANCE: We are developing our Chaperone-Assisted Crystallography (CAC) technology to solve the structure of protein systems of critical biomedical importance that have been recalcitrant to crystallization by traditional methods. A key innovation is our ability to rapidly generate designer antibodies that tightly bind the target molecules and promote the formation of high-quality crystals for x-ray crystallographic analysis. The technology will have a broad impact on the structural biology community and the structures determined with it will advance fundamental understanding of cellular functions and provide guidelines for drug development in a variety of diseases.