Genetically encodable epitopes to overcome size and resolution limits in cryo-EM

基因可编码表位可克服冷冻电镜中的尺寸和分辨率限制

• 批准号: 10017301
• 负责人: Jeremy Mills
• 金额: $ 23.48万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10017301
• 关键词: 3-Dimensional; Address; Adoption; Affinity; Amber; Amino Acids; Amino Acyl-tRNA Synthetases; Animals; Antibodies; Basic Science; Binding; Biological Models; Carbon; Chemical Structure; Chemicals; Complex; Consumption; Cryoelectron Microscopy; Data; Data Collection; Development; Disease; Electron Microscopy; Elements; Epitopes; Escherichia coli; Ferritin; Glean; Goals; Gold; Immunoglobulin Fragments; Incubated; Lead; Length; Light; Lysine; Mediating; Methods; Modeling; Molecular Weight; Nature; Negative Staining; Nicotine; Nuclear Magnetic Resonance; Pharmaceutical Preparations; Positioning Attribute; Process; Protein Region; Proteins; Reporting; Research; Research Personnel; Resolution; Resources; Sampling; Side; Site; Structure; Surface; System; Techniques; Technology; Terminator Codon; Testing; Time; Validation; Variant; Work; X-Ray Crystallography; alpha helix; base; beta pleated sheet; beta-Galactosidase; computerized data processing; cost; dalton; design; flexibility; improved; method development; molecular size; particle; protein complex; reconstruction; structural biology; tool

ABSTRACT Cryo-electron microscopy (cryo-EM) is revolutionizing the field of structural biology by providing advantages over long-standing and more frequently used techniques including x-ray crystallography and nuclear magnetic resonance. Recent technological advancements have begun to expand the number and types of proteins that can be characterized using cryo-EM; however, a major barrier to the widespread adoption of the technique still exists. Namely, an inverse correlation exists between the molecular weight of the target protein and the resolution that can be achieved by electron microscopy, thereby limiting the utility of the technique to very large proteins or protein complexes. At present, only proteins larger than ~100,000 Daltons routinely give rise to data with resolutions that rival those obtained using x-ray crystallography. Current approaches to circumvent this problem generally rely on increasing the physical bulk of the target protein, often by identifying proteins that specifically interact with the protein under study. A frequently employed method of achieving this is to evolve highly specific antibodies against the target protein, which are then bound to the target protein in the form of Fabs. While general, this method suffers from the drawbacks that new antibodies must be developed for each target protein, which often requires the use of animals and is time consuming and costly. Furthermore, no control over the site of Fab binding on the target is afforded using this method. Here, we propose to address this challenge by developing a single residue “epitope” in the form of a non-canonical amino acid (NCAA) that is specifically recognized by an existing antibody. Using the well- established amber stop codon suppression technology, NCAAs can be site-specifically incorporated at essentially any position in a target protein. Antibodies raised against the NCAA would then be expected to specifically bind a target protein in which a surface-exposed residue had been replaced with the NCAA. Because this approach decouples the epitope bound by the antibody from features of the target protein, it obviates the need to evolve a new antibody for each protein under study and also affords direct control over the region of the protein targeted by the Fab. We will begin to explore this possibility in two focused aims. We will first use a previously reported antibody against the drug nicotine to probe variants of the protein ferritin in which nicotine-containing NCAAs have been incorporated. We will use this model system to identify ideal chemical parameters of the nicotine containing NCAA that optimize Fab binding and create a rigid protein-protein interface. In a second aim, we will explore the generality of our approach in proteins other than ferritin and attempt to push the size limits of cryo-EM by applying our technique to very small proteins. We ultimately hope to generate a new toolkit for high resolution structure determination using cryo-EM that both removes existing limitations regarding the use of Fabs and also allows the use of this technique by resource-limited researchers.

摘要 冷冻电子显微镜（cryo-EM）是结构生物学领域的一场革命， 包括X射线晶体学和核磁共振在内的长期存在和更频繁使用的技术 共振最近的技术进步已经开始扩大蛋白质的数量和类型， 可以使用cryo-EM进行表征;然而，广泛采用该技术的主要障碍仍然是 存在.即，在目标蛋白质的分子量和分辨率之间存在逆相关性 这可以通过电子显微镜实现，从而限制了该技术对非常大的蛋白质或 蛋白质复合物目前，只有大于~ 100，000道尔顿的蛋白质通常会产生以下数据： 分辨率与使用X射线晶体学获得的分辨率相媲美。目前解决这一问题的办法 通常依赖于增加靶蛋白的物理体积，通常通过鉴定特异性地 与所研究的蛋白质相互作用。实现这一目标的一个常用方法是进化高度特异性 针对靶蛋白的抗体，其然后以Fab的形式与靶蛋白结合。而 通常这种方法的缺点是必须为每种靶蛋白开发新的抗体， 这通常需要使用动物，并且耗时且昂贵。此外，没有对网站的控制 使用该方法提供了结合在靶标上的Fab的量。 在这里，我们建议通过开发一个单一的残基“表位”的形式来解决这个挑战， 非典型氨基酸（NCAA），其被现有抗体特异性识别。利用这口井- 在已建立的琥珀终止密码子抑制技术中，NCAA可以在 基本上靶蛋白中的任何位置。针对NCAA提出的抗体预计将 特异性结合靶蛋白，其中表面暴露的残基已被NCAA取代。因为 这种方法从靶蛋白的特征中分离出抗体结合的表位， 需要为每种研究中的蛋白质进化一种新的抗体，并且还可以直接控制蛋白质的区域。 Fab所针对的蛋白质。我们将开始在两个重点目标中探索这种可能性。 我们将首先使用先前报道的抗尼古丁药物的抗体来探测铁蛋白的变体 其中含有尼古丁的NCAAs已被掺入。我们将使用这个模型系统来识别理想的 含有NCAA的尼古丁的化学参数，其优化Fab结合并产生刚性蛋白质-蛋白质 接口.在第二个目标中，我们将探索我们的方法在铁蛋白以外的蛋白质中的普遍性， 尝试通过将我们的技术应用于非常小的蛋白质来推动冷冻EM的尺寸限制。我们最终希望 使用cryo-EM生成用于高分辨率结构确定的新工具包， 本发明的优点在于消除了关于Fab使用的限制，并且还允许资源有限的研究人员使用该技术。