Chaperone Assisted Crystallography

描述(申请人提供)：我们已经开发并验证了一种名为“伴侣辅助结晶学”(CAC)的强大的高通量方法，该方法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平台发展到这样一个水平，即它的应用将通过使困难的问题成为常规问题和使“不可能”的问题成为可能，从而对结构生物界产生变革作用。CAC方法的基础是使用合成的抗体片段作为“结晶伴侣”，它与目标蛋白质或RNA实体特异性地紧密结合，从而促进结晶并提供阶段信息。构成CAC方法基础的技术突破是一种创新的组合文库设计，它使用一种“简化的遗传密码”来产生针对非常广泛的目标分子的高功能合成抗体片段。使用这种方法，我们已经产生了针对顽固靶标的伴侣，包括膜蛋白和功能RNA，促进了结晶和随后的结构确定。主要成果包括全长KCSA钾离子通道和第一组内含子的P4P6结构域的结构。为了进一步扩展CAC技术的能力，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)分别为膜蛋白质和核酸量身定做的新的“简化遗传密码”伴侣文库；ii)捕获目标分子的优先构象状态；iii)将伴侣定位于目标分子上特定的预定义区域；iv)产生瞬时大分子复合体(例如DNA-蛋白质相互作用)的伴侣并使其稳定。V)以FAB结合蛋白的形式存在的“辅助伴侣”，可以被设计来引入反常的原子类型以用于MAD相变和改变表面性质，以及诱导晶格的形成。将对照一组五个高影响的结构性问题对新的CAC增强措施的有效性进行评估，这些问题已证明使用传统方法是完全难以解决的。这些“模型系统”包括：1)HIV-1整合酶-DNA复合体，2)F-肌动蛋白四聚体，3)MERR转录因子家族，4)NaChBac Na+通道，5)构象锁定形式的22-肾上腺素受体。使用这些系统所获得的经验将用于进一步完善CAC技术。为了让结构生物界能够使用这项强大的技术，我们建立了一个校外项目，研究人员可以将他们顽固的蛋白质送到CAC管道，或者派遣人员到我们的实验室监督结晶伴侣的生产。与公共健康相关：我们正在开发我们的伴侣辅助结晶(CAC)技术，以解决传统方法难以结晶的具有关键生物医学意义的蛋白质系统的结构。一项关键的创新是我们能够快速产生设计抗体，紧密结合目标分子，并促进高质量晶体的形成，用于X射线结晶学分析。这项技术将对结构生物界产生广泛的影响，用它确定的结构将促进对细胞功能的基本了解，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.