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.

抽象的 冷冻电子显微镜（Cryo-EM）通过提供优势来彻底改变结构生物学领域 长期和更常用的技术，包括X射线晶体学和核磁力 谐振。最近的技术进步已经开始扩大蛋白质的数量和类型 可以使用冷冻EM来表征；但是，仍然是采用该技术的宽度的主要障碍 存在。也就是说，靶蛋白的分子量与分辨率之间存在逆相关性 可以通过电子显微镜来实现，从而将技术的效用限制为非常大的蛋白质或 蛋白质复合物。目前，只有大于约100,000 dalton的蛋白质通常会引起数据 有风险使用X射线晶体学获得的决议。当前解决这个问题的方法 通常依赖于增加靶蛋白的物理大量，通常是通过识别专门的蛋白质 与正在研究的蛋白质相互作用。实现这一目标的一种经常采用的方法是发展高度具体 抗靶蛋白的抗体，然后以Fab的形式与靶蛋白结合。尽管 一般，此方法遭受了必须为每种靶蛋白开发新抗体的缺点， 通常需要使用动物，并且耗时且昂贵。此外，无法控制网站 使用此方法提供了目标上的Fab绑定。 在这里，我们建议通过以某种形式开发单个居住的“表位”来应对这一挑战。 非典型氨基酸（NCAA）被现有抗体特别识别。使用井 建立的琥珀终止密码子抑制技术，NCAA可以特定于现场合并 本质上是目标蛋白中的任何位置。然后，针对NCAA提出的抗体将有望 特异性结合了靶蛋白，其中已被NCAA取代了表面暴露的住所。因为 这种方法将抗体结合的表位与靶蛋白的特征结合，它避免了 需要进化针对正在研究的每种蛋白质的新抗体，并且还可以直接控制 蛋白质是由Fab靶向的。我们将开始在两个重点目标中探索这种可能性。 我们将首先使用针对药物尼古丁的先前报道的抗体来探测蛋白铁蛋白的变体 其中已纳入了含尼古丁的NCAA。我们将使用此模型系统来识别理想 含有NCAA的尼古丁的化学参数，可优化FAB结合并创建刚性蛋白质蛋白质 界面。在第二个目标中，我们将探索除铁蛋白以外的蛋白质中我们方法的普遍性 尝试通过将我们的技术应用于非常小的蛋白质来推动Cryo-EM的尺寸限制。我们最终希望 使用Cryo-EM生成一个用于高分辨率结构确定的新工具包 有关使用Fab的限制，还允许使用有限的资源研究人员使用此技术。